Compositions comprising a combination of nivolumab and ipilimumab

ABSTRACT

This provides pharmaceutical compositions that comprise a combination of an anti-cancer agent which is an first antibody and a second antibody. In some embodiments, the first antibody is an anti-Programmed Death-1 (PD-1) antibody. In certain embodiments, the composition is a fixed dose formulation. In certain embodiments, the composition is administered as a flat-dose. The disclosure also provides a kit for treating a subject afflicted with a disease, the kit comprising a dosage of any composition disclosed herein and instructions for using the composition in any of the disclosed methods for treating a disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.15/130,513, filed on Apr. 14, 2016, which claims benefit to U.S.Provisional Application Nos. 62/303,855, filed Mar. 4, 2016; 62/269,000,filed Dec. 17, 2015; 62/265,268, filed Dec. 9, 2015; and 62/149,325,filed Apr. 17, 2015, which are incorporated herein by reference in theirentireties.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name:3338_0260005_SL.txt; Size: 16,858 bytes; and Date of Creation: Nov. 30,2016) is herein incorporated by reference in its entirety.

Throughout this application, various publications are referenced inparentheses by author name and date, or by patent number or patentPublication number. The disclosures of these publications are herebyincorporated in their entireties by reference into this application inorder to more fully describe the state of the art as known to thoseskilled therein as of the date of the invention described and claimedherein. However, the citation of a reference herein should not beconstrued as an acknowledgement that such reference is prior art to thepresent invention.

FIELD OF THE INVENTION

This invention relates to pharmaceutical compositions that comprise acombination of an immune checkpoint antibody and a second antibody at afixed dose formulation.

BACKGROUND OF THE INVENTION

Human cancers harbor numerous genetic and epigenetic alterations,generating neoantigens potentially recognizable by the immune system(Sjoblom et al. (2006) Science 314:268-74). The adaptive immune system,comprised of T and B lymphocytes, has powerful anti-cancer potential,with a broad capacity and exquisite specificity to respond to diversetumor antigens. Further, the immune system demonstrates considerableplasticity and a memory component. The successful harnessing of allthese attributes of the adaptive immune system would make immunotherapyunique among all cancer treatment modalities.

Recently, several immune checkpoint pathway inhibitors have begun toprovide new immunotherapeutic approaches for treating cancer, includingthe development of an antibody (Ab), ipilimumab (YERVOY®), that binds toand inhibits Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4) for the treatmentof patients with advanced melanoma and the development of antibodiessuch as nivolumab and pembrolizumab (formerly lambrolizumab; USANCouncil Statement (2013) Pembrolizumab: Statement on a nonproprietaryname adopted by the USAN Council (ZZ-165), Nov. 27, 2013) that bindspecifically to the Programmed Death-1 (PD-1) receptor and block theinhibitory PD-1/PD-1 ligand pathway.

Immune checkpoint antibodies can be administered in combination withother antibodies. Nonetheless, administering two antibodies can beburdensome due to different dosing and dosing interval between twoantibodies, thereby causing multiple intravenous injections at differenttime points. Furthermore, two antibodies can have drastically differentstability profiles. Due to the unique nature of each antibody, e.g.,variations in Fc glycosylation, partial heavy chain C-terminal Lysprocessing, Fc methionine oxidation, hinge-region cleavage, andglycation of Lys residues, each antibody has varied physicochemicaland/or thermodynamic properties, e.g., different degradation profileswhen exposed to heat, freezing, light, pH extremes, agitation,sheer-stress, some metals, and organic solvents. Therefore, while asingle formulation containing two antibodies would improve convenience,the unique nature of each antibody makes such a single formulationdifficult to identify.

SUMMARY OF THE INVENTION

The present invention relates to a pharmaceutical composition comprisingan X amount of a first antibody or an antigen-binding fragment thereof,which comprises an anti-PD-1 antibody or an antigen-binding fragmentthereof, and a Y amount of a second antibody or an antigen-bindingfragment thereof, wherein the ratio of the X amount to the Y amount isabout 50:1 to about 1:50. In some embodiments, the ratio of X to Y isabout 50:1, about 40:1, about 30:1, about 20:1, about 10:1, about 5:1,about 3:1, about 1:1, about 1:3, about 1:5, about 1:10, about 1:20,about 1:30, about 1:40, or about 1:50.

In some embodiments, the anti-PD-1 antibody is nivolumab orpembrolizumab. In a particular embodiment, the anti-PD-1 antibody isnivolumab.

In certain embodiments, the X amount of the first antibody or antigenbinding fragment thereof is at least about 60 mg, about 80 mg, about 100mg, about 120 mg, about 140 mg, about 160 mg, about 180 mg, about 200mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, or about 300mg. In some embodiments, the X amount of the first antibody is at leastabout 80 mg, about 160 mg, or about 240 mg. In other embodiments, the Xamount of the first antibody or antigen-binding fragment thereof isabout 60 mg, about 80 mg, about 100 mg, about 120 mg, about 140 mg,about 160 mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg,about 260 mg, about 280 mg, or about 300 mg. In certain embodiments, theX amount of the first antibody or antigen-binding fragment thereof isabout 80 mg or about 240 mg. In other embodiments, the X amount of thefirst antibody or antigen binding fragment thereof is more than at leastabout 300 mg. In some embodiments, the X amount of the first antibody orantigen binding fragment thereof is from at least about 300 mg to atleast about 500 mg, from at least about 300 mg to at least about 450 mg,from at least about 300 mg to at least about 400 mg, from at least about300 mg to at least about 350 mg, from at least about 350 mg to at leastabout 500 mg, from at least about 400 mg to at least about 500 mg, orfrom at least about 450 mg to at least about 500 mg. In certainembodiments, the X amount is at least about 300 mg, about 310 mg, about320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about470 mg, about 480 mg, about 490 mg, about 500 mg. In one particularembodiment, the X amount of the first antibody or antigen bindingfragment thereof is about 360 mg. In another embodiment, the X amount ofthe first antibody or antigen binding fragment thereof is about 480 mg.

In one aspect, the second antibody or antigen-binding fragment thereofcan be an anti-CTLA4 antibody. The ratio of the X amount of the firstantibody (e.g., an anti-PD-1 antibody or anti-PD-L1 antibody) to the Yamount of the second antibody (e.g., an anti-CTLA-4 antibody) is about3:1, about 1:1, or about 1:3. In some embodiments, (i) the X amount ofan anti-PD-1 antibody is about 240 mg and the Y amount of an anti-CTLA-4antibody is about 80 mg, (ii) the X amount is about 80 mg and the Yamount is about 80 mg; (iii) the X amount is about 160 mg and the Yamount is about 160 mg; (iv) the X amount is about 240 mg and the Yamount is about 240 mg; or (v) the X amount is about 80 mg and the Yamount is about 240 mg. In certain embodiments, the anti-CTLA4 antibodyis tremelimumab or ipilimumab.

In another aspect, the second antibody can be an anti-LAG3 antibody. Theratio of the X amount of the first antibody (e.g., an anti-PD-1 antibodyor anti-PD-L1 antibody) to the Y amount of the second antibody (e.g.,anti-LAG-3 antibody) is about 12:1, about 3:1, or about 1:1. In aparticular embodiment, the anti-LAG3 antibody is BMS-986016.

In other aspects, the second-antibody is an anti-CD137 antibody. In someembodiments, the ratio of the X amount of the first antibody (e.g., ananti-PD-1 antibody or anti-PD-L1 antibody) to the Y amount of the secondantibody (e.g., anti-CD-137 antibody) is about 1:1, about 1:2, about1:3, about 1:4, about 1:5, about 1:10, about 1:20, about 1:30, about1:40, about 1:50, about 50:1, about 40:1, about 30:1, about 20:1, about10:1, about 5:1, about 4:1 or about 2:1. In a particular embodiment, theanti-CD137 antibody is urelumab.

In some aspects, the second antibody is an anti-KIR antibody. In someembodiments, the ratio of the X amount of the first antibody (e.g., ananti-PD-1 antibody or anti-PD-L1 antibody) to the Y amount of the secondantibody (e.g., anti-KIR antibody) is about 30:1, about 10:1, about 3:1,about 1:1, or about 1:2. In some embodiments, the anti-KIR antibody is1-7F9 or lirilumab.

In certain aspects, the second antibody can be an anti-GITR antibody. Insome embodiments, the anti-GITR antibody is MK4166 or TRX518. In otherembodiments, the ratio of the X amount of the first antibody (e.g., ananti-PD-1 antibody or anti-PD-L1 antibody) to the Y amount of the secondantibody (e.g., anti-GITR antibody) is about 1:1, about 1:2, about 1:3,about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about1:10, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1,about 8:1, about 9:1, or about 10:1.

In other aspects, the second antibody is selected from the groupconsisting of: an anti-TGFβ antibody, an anti-IL-10 antibody, ananti-B7-H4 antibody, an anti-Fas ligand antibody, an anti-CXCR4antibody, an anti-mesothelin antibody, an anti-CD27 antibody, ananti-CD73 antibody, and any combination thereof.

A pharmaceutical composition of the invention can further comprises oneor more additional components selected from the group consisting of: abulking agent, a stabilizing agent, a chelating agent, a surfactant, abuffering agent, an ionic agent and any combination thereof.

In one embodiment, the pharmaceutical composition of the presentinvention is formulated in one or more of various buffering agents. Forexample, a composition of the invention can be formulated in a Tris-Cl,histidine, citrate or Tris-citrate buffer. In one embodiment, thecomposition is formulated in a Tris-Cl buffer, the concentration ofTris-Cl being at least about 5 mM, about 10 mM, about 15 mM, about 20mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, or about 50 mM.In another embodiment, the concentration of Tris-Cl is about 20 mM. Inother embodiments, the composition is formulated in a citrate buffer,the concentration of citrate being at least about 5 mM, about 10 mM,about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about40 mM, or about 50 mM. In a particular embodiment, the citrateconcentration is about 10 mM or about 20 mM. In some embodiments, thecomposition is formulated in a histidine buffer, the concentration ofhistidine being at least about 5 mM, about 10 mM, about 15 mM, about 20mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, or about 50 mM.In some embodiments, the histidine concentration is about 20 mM. Inother embodiments, the composition is formulated in a Tris-citratebuffer, the concentration of Tris-Cl being at least about 5 mM, about 10mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM,about 40 mM, or about 50 mM, and the concentration of citrate being atleast about 2 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM,about 25 mM, about 30 mM, about 35 mM, about 40 mM, or about 50 mM. Incertain embodiments, the concentration of Tris-Cl is about 13.3 mM andthe concentration of citrate is about 6.7 mM.

The composition of the invention can have a pH ranging from about 5 toabout 8. For example, the pH of the composition can be at least about 5,about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3,about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6,about 7.7, about 7.8, about 7.9, or about 8.0. In some embodiments, thepH of the composition is at least about 6.0, about 6.2, about 6.5, about6.6 or about 7.0

The composition can further comprise a bulking agent. In certainembodiments, the bulking agent is selected from the group consisting ofNaCl, mannitol, glycine, alanine, and any combination thereof.

In certain embodiments, the composition comprises a stabilizing agent.The stabilizing agent is selected from the group consisting of sucrose,trehalose, raffinose, arginine; or any combination thereof.

In other embodiments, the composition comprises a chelating agent. Thechelating agent is selected from the group consisting ofdiethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraaceticacid, nitrilotriacetic acid, and any combination thereof.

In certain embodiments, the surfactant is selected from the groupconsisting of polysorbate 80 (PS80), polysorbate 20 (PS20), and anycombination thereof.

In some embodiments, the composition comprises NaCl at a concentrationof at least about 5 mM, at least about 10 mM, at least about 15 mM, atleast about 20 mM, at least about 25 mM, at least about 30 mM, at leastabout 35 mM, at least about 40 mM, at least about 45 mM, at least about50 mM, at least about 60 mM, at least about 70 mM, at least about 75 mM,at least about 80 mM, at least about 90 mM, at least about 100 mM, atleast about 110 mM, at least about 120 mM, at least about 130 mM, atleast about 140 mM, at least about 150 mM, at least about 175 mM, atleast about 200 mM, at least about 225 mM, at least about 250 mM, atleast about 275 mM, at least about 300 mM, at least about 350 mM, atleast about 400 mM, at least about 450 mM or at least about 450 mM. Insome embodiments, the concentration of NaCl is about 100 mM, about 96.15mM, about 83.3 mM, about 78.57 mM or about 50 mM.

In some embodiments, the composition comprises mannitol (% w/v) USP at aconcentration of at least about 0.25%, at least about 0.5%, at leastabout 0.75%, at least about 1%, at least about 1.5%, at least about 2%,at least about 2.5%, at least about 3%, at least about 3.5%, at leastabout 4%, at least about 4.5%, at least about 5%, at least about 7.5% orat least about 10%. In some embodiments, the concentration of mannitolis about 1%, about 1.15%, about 1.67%, about 1.86%, or about 3%.

In some embodiments, the composition comprises DTPA, USP at aconcentration of at least about 5 μM, at least about 10 μM, at leastabout 15 μM, at least about 20 μM, at least about 25 μM, at least about30 μM, at least about 40 μM, at least about 50 μM, at least about 60 μM,at least about 70 μM, at least about 75 μM, at least about 80 μM, atleast about 90 μM, at least about 100 μM, at least about 110 μM, atleast about 120 μM, at least about 130 μM, at least about 140 μM, atleast about 150 μM, at least about 175 μM, or at least about 200 μM. Insome embodiments, the concentration of DTPA is about 20 μM, about 50 μM,about 65.71 μM, about 73.3 μM, about 93.85 μM, or 100 μM.

In some embodiments, the composition comprises PS80 (% w/v) at aconcentration of at least about 0.005%, at least about 0.01%, at leastabout 0.015%, at least about 0.02%, at least about 0.03%, at least about0.04%, at least about 0.05%, at least about 0.06%, at least about 0.07%,at least about 0.08%, at least about 0.09%, or at least about 0.1%. Insome embodiments, the concentration of PS80 is about 0.01%, about0.012%, about 0.013%, about 0.02%, about 0.23%, about 0.04%, or about0.05%.

In some embodiments, the composition comprises sucrose (% w/v) at aconcentration of at least about 1%, at least about 2%, at least about3%, at least about 4%, at least about 4.5%, at least about 5%, at leastabout 5.5%, at least about 6%, at least about 6.5%, at least about 7%,at least about 7.5%, at least about 8%, at least about 8.5%, at leastabout 9%, at least about 9.5%, at least about 10%, at least about 12% orat least about 15%. In some embodiments, the concentration of sucrose isabout 6% or about 8.5%.

In certain embodiments, the invention includes the followingcompositions: (i) a pharmaceutical composition comprising a 1:1 ratio ofnivolumab to ipilimumab in a buffer comprising about 13.3 mM Tris, about6.7 mM citrate, about 1.67% mannitol, about 83.3 mM NaCl, about 73.3 μMDTPA and about 0.013% PS80 at a pH of about 6.2; (ii) a pharmaceuticalcomposition comprising a 3:1 ratio of nivolumab to ipilimumab in aTris-citrate buffer comprising about 1.15% mannitol, about 96.15 mMNaCl, about 93.85 μM DTPA and about 0.012% PS80 at a pH of about 6.6;(iii) a pharmaceutical composition comprising a 1:3 ratio of nivolumabto ipilimumab in a Tris-citrate buffer comprising about 1.86% mannitol,about 78.57 mM NaCl, about 65.71 μM DTPA and about 0.023% PS80 at a pHof about 6.0; (iv) a pharmaceutical composition comprising a 3:1 ratioof nivolumab to ipilimumab in a 20 mM histidine buffer comprising about50 mM NaCl, about 50 μM DTPA, about 6% sucrose, and about 0.05% PS80 atabout pH 6; (v) a pharmaceutical composition comprising a 3:1 ratio ofnivolumab to ipilimumab in a about 20 mM histidine buffer comprisingabout 50 mM NaCl, about 50 μM DTPA, about 6% sucrose, and about 0.05%PS80 at about pH 7; (vi) a pharmaceutical composition comprising a 3:1ratio of nivolumab to ipilimumab in an about 20 mM histidine buffercomprising about 50 μM DTPA, about 8.5% sucrose, and about 0.05% PS80 atabout pH 6; (vii) a pharmaceutical composition comprising comprises a3:1 ratio of nivolumab to ipilimumab in an about 20 mM citrate buffercomprising about 50 mM NaCl, about 50 μM DTPA, about 6% sucrose, andabout 0.05% PS80 at about pH 6; (viii) a pharmaceutical compositioncomprising a 3:1 ratio of nivolumab to ipilimumab in an about 20 mMcitrate buffer comprising about 50 mM NaCl, about 20 μM DTPA, about 3%mannitol, and about 0.04% PS80 at about pH 6; (ix) a pharmaceuticalcomposition comprising a 1:1 ratio of nivolumab to ipilimumab in anabout 20 mM citrate buffer comprising about 50 mM NaCl, about 100 μMDTPA, about 3% mannitol, and about 0.02% PS80 at about pH 6; (x) apharmaceutical composition comprising a 1:1 ratio of nivolumab toipilimumab in an about 20 mM citrate buffer comprising about 50 mM NaCl,about 100 μM DTPA, about 3% mannitol, and about 0.02% PS80 at about pH6.5; (xi) a pharmaceutical composition comprising a 1:1 ratio ofnivolumab to ipilimumab in an about 20 mM citrate buffer comprisingabout 100 mM NaCl, about 100 μM DTPA, about 1.0% mannitol, and about0.02% PS80 at about pH 6.5; or (xii) a pharmaceutical compositioncomprising a 1:1 ratio of nivolumab to ipilimumab in an about 20 mMcitrate buffer comprising about 50 mM NaCl, about 100 μM DTPA, about 6%sucrose, and about 0.02% PS80 at about pH 6.0.

In other embodiments, the invention includes a pharmaceuticalcomposition comprising a 1:3 ratio of nivolumab to ipilimumab comprisingabout 4.62 mg/ml nivolumab, about 1.54 mg/ml ipilimumab, about 18.5 mMTris Hydrochloride, about 1.5 mM Sodium Citrate Dihydrate, about 96.2 mMNaCl, about 1.2% Mannitol, about 93.9 μM Pentetic Acid, and about 0.012%PS80 at about pH 6.0 or a pharmaceutical composition comprising a 1:3ratio of nivolumab to ipilimumab comprising about 4.61 mg/ml nivolumab,about 1.54 mg/ml ipilimumab, about 18.46 mM Tris Hydrochloride, about1.54 mM Sodium Citrate Dihydrate, about 96.15 mM NaCl, about 1.15%Mannitol, about 93.85 μM Pentetic Acid, and about 0.012% PS80 at aboutpH 6.3.

The composition of the invention after formulation can be stable andstored at about 5° C. for at least about 1 week, at least about 2 weeks,at least about 1 month, at least about 2 months, at least about 3months, at least about 6 months, at least about 9 months, at least about1 year, at least about 2 years or at least about 5 years. In someembodiments, the composition is stable and can be stored at about 40° C.for at least about 1 week, at least about 2 weeks, at least about 1month, at least about 2 months, at least about 3 months, at least about6 months, at least about 9 months, at least about 1 year, at least about2 years or at least about 5 years. In other embodiments, the compositionis stable and can be stored at about 25° C. for at least about 1 week,at least about 2 weeks, at least about 1 month, at least about 2 months,at least about 3 months, at least about 6 months, at least about 9months, at least about 1 year, at least about 2 years or at least about5 years.

The pharmaceutical composition of the invention can exhibit a minimalchange of an acidic peak upon stress, e.g., after being stored at aparticular temperature for a long period of time. In one embodiment, thecomposition exhibits a change of an acidic peak less than about 10%,about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%,about 2%, or about 1% after being stored for about 6 months or about 3months at about 5° C. In certain embodiments, the composition exhibits achange of an acidic peak that is less than about 15%, about 14%, about13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%,about 6%, about 5%, about 4%, about 3%, about 2% or about 1% after beingstored for about 3 months at about 25° C. In other embodiments, thecomposition exhibits a change of an acidic peak that is less than about15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%,about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2% orabout 1% after being stored for about 3 months at about 40° C.

The composition of the invention can also exhibit a minimal change ofhigh molecular weight peak after being stored for a long period of time,several weeks, months, or years. In one embodiment, the compositionexhibits a change of a high molecular weight peak less than about 5%,about 4%, about 3%, about 2%, about 1.5%, about 1.4%, about 1.3%, about1.2%, about 1.1%, about 1%, about 0.9%, about 0.8%, about 0.7%, about0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1%after being stored for about 3 months at about 4° C. In anotherembodiment, the composition exhibits a change of a high molecular weightpeak that is less than about 5%, about 4%, about 3%, about 2%, about1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1%, about0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about0.3%, about 0.2%, or about 0.1% after being stored for about 2 months orabout 3 months at about 25° C. In other embodiments, the compositionexhibits a change of a high molecular weight peak that is less thanabout 5%, about 4%, about 3%, about 2%, about 1.5%, about 1.4%, about1.3%, about 1.2%, about 1.1%, about 1%, about 0.9%, about 0.8%, about0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, orabout 0.1% after being stored for about 2 months or about 3 months atabout 40° C.

Furthermore, in certain embodiments, the composition can exhibit aminimal change of a main peak determined by Capillary IsoelectricFocusing (cIEF) analysis. In one embodiment, the composition shows achange of a main peak less than about 5%, about 4%, about 3%, about 2%,about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1%,about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%,about 0.3%, about 0.2%, or about 0.1% after being stored for about 1month at about 4° C. In some embodiments, the composition exhibits achange of a main peak of Capillary Isoelectric Focusing (cIEF) analysisthat is less than about 5%, about 4%, about 3%, about 2%, about 1.5%,about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1%, about 0.9%,about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%,about 0.2%, or about 0.1% after being stored for about 1 month at about25° C. In some embodiments, the composition exhibits a change of a mainpeak of Capillary Isoelectric Focusing (cIEF) analysis that is less thanabout 5%, about 4%, about 3%, about 2%, about 1.5%, about 1.4%, about1.3%, about 1.2%, about 1.1%, about 1%, about 0.9%, about 0.8%, about0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, orabout 0.1% after being stored for about 1 month at about 40° C.

In some embodiments, the composition exhibits a minimal change of a lowmolecular weight peak. In one embodiment, the composition exhibits achange of a low molecular weight peak less than about 5%, about 4%,about 3%, about 2%, about 1.5%, about 1.4%, about 1.3%, about 1.2%,about 1.1%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%,about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1% afterbeing stored for about 2 months at about 40° C. In certain embodiments,the composition exhibits a change of a low molecular weight peak that isless than about 5%, about 4%, about 3%, about 2%, about 1.5%, about1.4%, about 1.3%, about 1.2%, about 1.1%, about 1%, about 0.9%, about0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about0.2%, or about 0.1% after being stored for about 2 months at about 25°C. In certain embodiments, the composition exhibits a change of a lowmolecular weight peak that is less than about 5%, about 4%, about 3%,about 2%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%,about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%,about 0.4%, about 0.3%, about 0.2%, or about 0.1% after being stored forabout 2 months at about 4° C.

In some embodiments, the composition is diluted prior to use. In someembodiments, the composition is diluted with 0.9% Sodium ChlorideInjection, USP or 5% Dextrose Injection, USP prior to use. In someembodiments, the composition is diluted to obtain a desiredconcentration of first and second antibody.

In some embodiments, the disclosure is directed to a kit comprising acomposition disclosed herein.

In some embodiments, the disclosure is directed to method of making acomposition disclosed herein. In some embodiments, a formulationcomprising the anti-PD-1 antibody drug product is mixed with aformulation comprising the second antibody drug product to obtain thedesired ratio in a final drug product with no buffer changes. In someembodiments, a formulation comprising the anti-PD-1 antibody drugsubstance and a formulation comprising the second antibody drugsubstance is subject to buffer exchanges and/or concentration beforebeing mixed to obtain the desired ratio in a final drug product.

In some embodiments, the disclosure is directed to a method ofmodulating an immune response to a patient in need thereof comprisingadministering a composition disclosed herein to the patient.

In some embodiments, the disclosure is directed to a method ofadministering two antibodies at the same time to a patient in needthereof comprising administering to the patient a composition disclosedherein, wherein the antibodies are capable of treating at least onedisease or condition.

In some embodiments, the disclosure is directed to method of treating adisease or condition comprising administering a composition disclosedherein to a patient.

In some embodiments, the disease or condition is an infectious disease.In some embodiments, the disease is cancer. In some embodiments, thecancer is melanoma cancer, renal cancer, prostate cancer, breast cancer,colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer,cancer of the head or neck, cutaneous or intraocular malignant melanoma,uterine cancer, ovarian cancer, rectal cancer, cancer of the analregion, stomach cancer, testicular cancer, uterine cancer, carcinoma ofthe fallopian tubes, carcinoma of the endometrium, carcinoma of thecervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin'sDisease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of thesmall intestine, cancer of the endocrine system, cancer of the thyroidgland, cancer of the parathyroid gland, cancer of the adrenal gland,sarcoma of soft tissue, cancer of the urethra, cancer of the penis,chronic or acute leukemias including acute myeloid leukemia, chronicmyeloid leukemia, acute lymphoblastic leukemia, chronic lymphocyticleukemia, solid tumors of childhood, lymphocytic lymphoma, cancer of thebladder, cancer of the kidney or ureter, carcinoma of the renal pelvis,neoplasm of the central nervous system (CNS), primary CNS lymphoma,tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitaryadenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer,T-cell lymphoma, environmentally induced cancers including those inducedby asbestos, and any combinations thereof.

In some embodiments, the composition is administered intravenously. Insome embodiments, the composition is diluted prior to administration. Insome embodiments, the composition is administered at a flat dose. Insome embodiments, the amount of the first antibody and the amount of thesecond antibody administered to the patient at a single dose areidentical the X amount and the Y amount, respectively. In someembodiments, the composition is administered at a weight-based dose. Insome embodiments, the amount of the first antibody administered to thepatient is at least about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg,about 2 mg/kg, about 3 mg/kg or about 5 mg/kg. In some embodiments, theamount of the first antibody administered to the patient at least about1 mg/kg.

In some embodiments, the composition is administered at least aboutweekly, at least about twice weekly, at least about every two weeks, atleast about every three weeks, or at least about monthly. In someembodiments, the administering lasts for at least about 8 weeks, atleast about 12 weeks, at least about 3 months, at least about 6 months,at least about 9 months, at least about 1 year, at least about 18months, at least about 2 years or greater than 2 years. In someembodiments, the patient is also treated with another anti-cancer agent.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the compositions of an anti-CTLA4 antibody (i.e.,ipilimumab) and an anti-PD-1 antibody (i.e., nivolumab) drug substance(DS) and drug product (DP) formulations.

FIG. 2A shows the results of size exclusion chromatography (SEC) of thecombined 1:1 ratio fixed dosing combination (FDRC=fixed dose ratiocombination) formulation of an anti-PD-1 antibody (e.g., nivolumab) andan anti-CTLA-4 antibody (e.g., ipilimumab). FIG. 2B shows imagedcapillary isoelectric focusing (cIEF) analyses of the combined 1:1 ratiofixed dosing combination (FDRC=fixed dose ratio combination) formulationof an anti-PD-1 antibody (e.g., nivolumab) and an anti-CTLA-4 antibody(e.g., ipilimumab). EC indicates a mix of Tris and citrate buffers. Thenivolumab DP and ipilimumab DP results are shown as controls; and thecombination of an anti-PD-1 antibody and an anti-CTLA4 antibody (e.g.,nivolumab and ipilimumab) are shown as EC FDRC in FIGS. 2A-B. FIG. 2Ashows the actual change in the high molecular weight (HMW) peak size (%)in formulations stored for 3 months at 40° C. as compared to day-0controls. The adjusted pH at 25° C. and the concentrations ofpolysorbate 80 (PS80), NaCl, and Mannitol for each prototype formulationare shown below the x-axis in FIG. 2A. FIG. 2B shows the actual changein the acidic peak size (%) of nivolumab and ipilimumab in formulationsstored for 6 months at 5° C. as compared to day-0 controls. The adjustedpH at 25° C. and the concentrations of NaCl and Mannitol for eachformulation are shown below the x-axis in FIG. 2B. Data points arelabeled as N (nivolumab), C (combination of nivolumab and ipilimumab),and I (ipilimumab) in FIGS. 2A-B.

FIGS. 3A-B show the results of SEC analyses of the 1:3, 1:1, or 3:1ratio fixed dosing formulations for an anti-PD-1 antibody (e.g.,nivolumab) and an anti-CTLA-4 antibody (e.g., ipilimumab). The nivolumabDP and ipilimumab DP results are shown as controls; and the combinationof an anti-PD-1 antibody and an anti-CTLA4 antibody (e.g., nivolumab andipilimumab) are shown as EC (FIGS. 3A-B). FIG. 3A shows the HMW peaksize (%) at day 0 (Initial), the HMW peak size after 2 months at 40° C.,and the change in HMW peak size between the day-0 control and theformulation at 2 months at 40° C. for each sample and prototype (EC: pH6.0 (1 ipilimumab:3 nivolumab); EC: pH 6.2 (1 ipilimumab:1 nivolumab);and EC: pH 6.6 (3 ipilimumab:1 nivolumab)) formulation. FIG. 3B showsthe LMW peak size (%) at day 0 (Initial), the LMW peak size after 2months at 40° C., the change in LMW peak size between the day-0 controland each formulation following storage for 2 months at 40° C., and thechange in LMW peak size between the day-0 control and each formulationfollowing storage for 3 months at 25° C. The concentrations of theadjusted pH at 25° C., ipilimumab concentration, nivolumabconcentration, PS80, and NaCl for each formulation are shown below thex-axes (FIGS. 3A-B).

FIGS. 4A-C show the results of cIEF analysis of the 1:3, 1:1, or 3:1ratio fixed dosing formulations for an anti-PD-1 antibody (e.g.,nivolumab) and an anti-CTLA-4 antibody (e.g., ipilimumab) followingstorage for 3 months at 25° C. (FIG. 4A), 3 months at 5° C. (FIG. 4B),and 1 month at 25° C. (FIG. 4C). The nivolumab DP and ipilimumab DPresults are shown as controls (FIGS. 4A-C). The actual differences inthe nivolumab (N) and ipilimumab (I) acidic peak sizes (%) at theselected time points relative to the day-0 controls are shown for thecontrols and each prototype (EC: pH 6.0 (1 ipilimumab:3 nivolumab); EC:pH 6.2 (1 ipilimumab:1 nivolumab); and EC: pH 6.6 (3 ipilimumab:3nivolumab)) formulation (FIGS. 4A-C). The theoretical pH at 25° C., thebuffer type, and the ratio of ipilimumab to nivolumab for eachformulation are shown below the x-axis in FIGS. 4A-B; and the NaClconcentration, the theoretical pH at 25° C., and the ratio of ipilimumabto nivolumab for each formulation are shown below the x-axis in FIG. 4C.The theoretical pH is equivalent to the stability study pH (FIGS. 4A-C).

FIGS. 5A-B show the results of SEC (FIG. 5A) and cIEF (FIG. 5B) analysesof the novel design-of-experiment (DoE) 3:1 Ratio Fixed Dosingformulations for an anti-PD-1 antibody (e.g., nivolumab) and ananti-CTLA-4 antibody (e.g., ipilimumab). The nivolumab DP and ipilimumabDP results are shown as controls (FIGS. 5A-B). FIG. 5A shows the HMWpeak size (%) at day 0 (Initial), the HMW peak size after 3 months at40° C., the change in HMW peak size between the day-0 control and theformulation following storage for 3 months at 40° C., and the change inHMW peak size between the day-0 control and the formulation followingstorage for 3 months at 25° C. for the control formulations and eachprototype formulation (Combo New, Combo 4, Combo 5, Combo 6, and Combo8). FIG. 5B shows the actual differences in the nivolumab (N) andipilimumab (I) acidic peak sizes (%) relative to the day-0 controls foreach formulation prototype following storage for 3 months at 25° C. Theratio of ipilimumab to nivolumab; the concentrations of NaCl, mannitol,and sucrose; the theoretical pH at 25° C., and the buffer type for eachformulation are shown below the x-axes (FIGS. 5A-B).

FIGS. 6A-B show the results of SEC analyses of the platform combined(PC) 1:1; 1:3; or 3:1 ratio fixed dosing formulations for an anti-PD-1antibody (e.g., nivolumab) and an anti-CTLA-4 antibody (e.g.,ipilimumab) following storage for 3 months at 40° C. (FIG. 6A) and 3months at 5° C. (FIG. 6B). The nivolumab DP and ipilimumab DP resultsare shown as controls (FIGS. 6A-B). FIG. 6A shows the actual change inthe HMW peak size (%) between the day-0 controls and each control andprototype (PC: pH 6.0-1:1; PC: pH 5.5-1:3; PC: pH 6.0-1:3; PC: pH6.5-1:3; and PC: pH 6.0-3:1) formulation following storage for 3 monthsat 40° C. FIG. 6B shows the HMW peak size (%) at day 0 (Initial) and theHMW peak size after 3 months at 5° C. for each formulation. The buffertype and the ratio of ipilimumab to nivolumab for each formulation areshown below the x-axes (FIGS. 6A-B).

FIGS. 7A-B show the results of cIEF analyses of the platform combined(PC) 1:1; 1:3; or 3:1 ratio fixed dosing formulations for an anti-PD-1antibody (e.g., nivolumab) and an anti-CTLA-4 antibody (e.g.,ipilimumab) following storage for 3 months at 25° C. (FIG. 7A) and 3months at 5° C. (FIG. 7B). The nivolumab DP and ipilimumab DP resultsare shown as controls (FIGS. 7A-B). FIG. 7A shows the actual differencesin the nivolumab (N) and ipilimumab (I) acidic peak sizes (%) relativeto the day-0 controls for each control and prototype (PC: pH 6.0-1:1;PC: pH 5.5-1:3; PC: pH 6.0-1:3; PC: pH 6.5-1:3; and PC: pH 6.0-3:1)formulation following storage for 3 months at 25° C. FIG. 7B shows theactual differences in the nivolumab (N) and ipilimumab (I) acidic peaksizes (%) relative to the day-0 controls for each formulation followingstorage for 3 months at 5° C. The buffer type and the ratio ofipilimumab to nivolumab for each formulation are shown below the x-axes(FIGS. 7A-B).

FIG. 8 shows the results of SEC analyses of the nivolumab-DP-based FDRC(1:1) formulations following storage for 1 month at 40° C. The nivolumabDP and ipilimumab DP results are shown as controls (FIG. 8). The actualchange in the BMW peak size (%) between the day-0 controls and theformulations following storage for 1 month at 40° C. is shown for eachcontrol and prototype (A, B, C, and D) formulation (FIG. 8). The ratioof ipilimumab to nivolumab, the buffer type, and the theoreticaladjusted pH for each formulation are shown below the x-axis (FIG. 8).

FIG. 9 shows the results of cIEF analyses of the nivolumab-DP-based FDRC(1:1) formulations following storage for 3 months at 25° C. Thenivolumab DP and ipilimumab DP results are shown as controls. FIG. 9shows the actual differences in the nivolumab (N) and ipilimumab (I)acidic peak sizes (%) relative to the day-0 controls for each controland prototype (A, B, C, and D) formulation following storage for 3months at 25° C. The ratio of ipilimumab to nivolumab, the buffer type,and the theoretical adjusted pH for each formulation are shown below thex-axis.

FIG. 10 shows the ipilimumab acidic peak degradation rate in the FDRCand commercial composition at 25° C./60% RH (relative humidity). TheFDRC composition is shown at Table 7.

FIG. 11 shows the nivolumab acidic peak degradation rate in the FDRC andcommercial composition at 25° C./60% RH. The FDRC composition is shownat Table 7.

FIG. 12 shows the acidic peak profile at 25° C. for ipilimumab andnivolumab in a pH ranging study.

FIG. 13 shows the size exclusion chromatography high molecular weightprofile of DP prototypes from the ruggedness study. The HMW profile ofthe FDRC DP remained unchanged after 6 months of storage at 2-8° C. and25° C.

FIG. 14 shows the size exclusion chromatography monomer profile of theFDRC DP after 6 months of storage at 2-8° C. and 25° C.

FIG. 15 shows the ipilimumab acidic peak profile. The evaluationindicates a pH dependence of deamidation at accelerated temperature of25° C., as indicated by an increase in acidic peak profile at highertemperature range of pH 7.0.

FIG. 16 shows the nivolumab acidic peak profile. The evaluationindicates a pH dependence of deamidation at accelerated temperature of25° C., as indicated by an increase in acidic peak profile at highertemperature range of pH 7.0.

FIG. 17 shows the ipilimumab main peak profile. The evaluation indicatesa pH dependence of deamidation at accelerated temperature of 25° C., asindicated by an increase in acidic peak profile at higher temperaturerange of pH 7.0.

FIG. 18 shows the nivolumab main peak profile. The evaluation indicatesa pH dependence of deamidation at accelerated temperature of 25° C., asindicated by an increase in acidic peak profile at higher temperaturerange of pH 7.0.

FIG. 19 shows the impact of pH on the cIEF profile.

FIG. 20 shows the iCIEF profile over a pH range of 5.4-6.6.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates pharmaceutical compositions comprisingboth an anti-PD-1 antibody and a second antibody. In some embodiments,the composition is a fixed dose formulation. The advantages of such asingle-formulation fixed dose composition can include improved medicalcompliance by reducing the time of treatment (for a composition given,for example, intravenously) or reduced administration burden (e.g.,multiple i.v. injections) and the ability to have a combined drugprofile for both drugs. However, such a single-formulation fixed dosecomposition can induce undesirable interactions between the twoantibodies, thereby reducing the total amount of active ingredient, aswell as a limitation on the ability of a physician to customize doses.

Terms

In order that the present disclosure may be more readily understood,certain terms are first defined. As used in this application, except asotherwise expressly provided herein, each of the following terms shallhave the meaning set forth below. Additional definitions are set forththroughout the application.

The term “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; Aand C; A and B; B and C; A (alone); B (alone); and C (alone).

It is understood that wherever aspects are described herein with thelanguage “comprising,” otherwise analogous aspects described in terms of“consisting of” and/or “consisting essentially of” are also provided.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Units, prefixes, and symbols are denoted in their Système Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. The headings provided herein are notlimitations of the various aspects of the disclosure, which can be hadby reference to the specification as a whole. Accordingly, the termsdefined immediately below are more fully defined by reference to thespecification in its entirety.

“Administering” refers to the physical introduction of a compositioncomprising a therapeutic agent to a subject, using any of the variousmethods and delivery systems known to those skilled in the art.Preferred routes of administration for the formulations disclosed hereininclude intravenous, intramuscular, subcutaneous, intraperitoneal,spinal or other parenteral routes of administration, for example byinjection or infusion. The phrase “parenteral administration” as usedherein means modes of administration other than enteral and topicaladministration, usually by injection, and includes, without limitation,intravenous, intramuscular, intraarterial, intrathecal, intralymphatic,intralesional, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion, as well as in vivo electroporation.In some embodiments, the formulation is administered via anon-parenteral route, preferably orally. Other non-parenteral routesinclude a topical, epidermal or mucosal route of administration, forexample, intranasally, vaginally, rectally, sublingually or topically.Administering can also be performed, for example, once, a plurality oftimes, and/or over one or more extended periods.

An “adverse event” (AE) as used herein is any unfavorable and generallyunintended or undesirable sign (including an abnormal laboratoryfinding), symptom, or disease associated with the use of a medicaltreatment. For example, an adverse event may be associated withactivation of the immune system or expansion of immune system cells(e.g., T cells) in response to a treatment. A medical treatment may haveone or more associated AEs and each AE may have the same or differentlevel of severity. Reference to methods capable of “altering adverseevents” means a treatment regime that decreases the incidence and/orseverity of one or more AEs associated with the use of a differenttreatment regime.

An “antibody” (Ab) shall include, without limitation, a glycoproteinimmunoglobulin which binds specifically to an antigen and comprises atleast two heavy (H) chains and two light (L) chains interconnected bydisulfide bonds, or an antigen-binding portion thereof. Each H chaincomprises a heavy chain variable region (abbreviated herein as V_(H))and a heavy chain constant region. The heavy chain constant regioncomprises three constant domains, C_(H1), C_(H2) and C_(H3). Each lightchain comprises a light chain variable region (abbreviated herein asV_(L)) and a light chain constant region. The light chain constantregion is comprises one constant domain, C_(L). The V_(H) and V_(L)regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDRs), interspersed withregions that are more conserved, termed framework regions (FR). EachV_(H) and V_(L) comprises three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (C1q)of the classical complement system.

An immunoglobulin may derive from any of the commonly known isotypes,including but not limited to IgA, secretory IgA, IgG and IgM. IgGsubclasses are also well known to those in the art and include but arenot limited to human IgG1, IgG2, IgG3 and IgG4. “Isotype” refers to theantibody class or subclass (e.g., IgM or IgG1) that is encoded by theheavy chain constant region genes. The term “antibody” includes, by wayof example, both naturally occurring and non-naturally occurringantibodies; monoclonal and polyclonal antibodies; chimeric and humanizedantibodies; human or nonhuman antibodies; wholly synthetic antibodies;and single chain antibodies. A nonhuman antibody may be humanized byrecombinant methods to reduce its immunogenicity in man. Where notexpressly stated, and unless the context indicates otherwise, the term“antibody” also includes an antigen-binding fragment or anantigen-binding portion of any of the aforementioned immunoglobulins,and includes a monovalent and a divalent fragment or portion, and asingle chain antibody.

The term “monoclonal antibody” (“mAb”) refers to a non-naturallyoccurring preparation of antibody molecules of single molecularcomposition, i.e., antibody molecules whose primary sequences areessentially identical, and which exhibits a single binding specificityand affinity for a particular epitope. A mAb is an example of anisolated antibody. MAbs may be produced by hybridoma, recombinant,transgenic or other techniques known to those skilled in the art.

A “human” antibody (HuMAb) refers to an antibody having variable regionsin which both the framework and CDR regions are derived from humangermline immunoglobulin sequences. Furthermore, if the antibody containsa constant region, the constant region is also derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody,” as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences. The terms “human” antibodies and “fully human”antibodies and are used synonymously.

A “humanized antibody” refers to an antibody in which some, most or allof the amino acids outside the CDR domains of a non-human antibody arereplaced with corresponding amino acids derived from humanimmunoglobulins. In one embodiment of a humanized form of an Ab, some,most or all of the amino acids outside the CDR domains have beenreplaced with amino acids from human immunoglobulins, whereas some, mostor all amino acids within one or more CDR regions are unchanged. Smalladditions, deletions, insertions, substitutions or modifications ofamino acids are permissible as long as they do not abrogate the abilityof the antibody to bind to a particular antigen. A “humanized” antibodyretains an antigenic specificity similar to that of the originalantibody.

A “chimeric antibody” refers to an antibody in which the variableregions are derived from one species and the constant regions arederived from another species, such as an antibody in which the variableregions are derived from a mouse antibody and the constant regions arederived from a human antibody.

An “anti-antigen” antibody refers to an antibody that binds specificallyto the antigen. For example, an anti-PD-1 antibody binds specifically toPD-1 and an anti-CTLA-4 antibody binds specifically to CTLA-4.

An “antigen-binding portion” of an antibody (also called an“antigen-binding fragment”) refers to one or more fragments of anantibody that retain the ability to bind specifically to the antigenbound by the whole antibody.

A “cancer” refers a broad group of various diseases characterized by theuncontrolled growth of abnormal cells in the body. Unregulated celldivision and growth divide and grow results in the formation ofmalignant tumors that invade neighboring tissues and may alsometastasize to distant parts of the body through the lymphatic system orbloodstream. A “cancer” or “cancer tissue” can include a tumor.

“CD137”, “CD-137”, “tumor necrosis factor receptor superfamily member 9(TNFRSF9)”, “4-1BB” and “induced by lymphocyte activation (ILA)” allrefer to the same member of the tumor necrosis factor receptor family.One activity CD137 has been implicated in is costimulatory activity foractivated T cells. (Jang et al. (1998) Biochem. Biophys. Res. Commun.242 (3): 613-20). The term “CD137” as used herein includes human CD137(hCTLA-4), variants, isoforms, and species homologs of hCD137, andanalogs having at least one common epitope with hCD137. The amino acidsequence for hCD137 can be found under GenBank Accession No. NP_001552.

“Cytotoxic T-Lymphocyte Antigen-4” (CTLA-4) refers to animmunoinhibitory receptor belonging to the CD28 family. CTLA-4 isexpressed exclusively on T cells in vivo, and binds to two ligands, CD80and CD86 (also called B7-1 and B7-2, respectively). The term “CTLA-4” asused herein includes human CTLA-4 (hCTLA-4), variants, isoforms, andspecies homologs of hCTLA-4, and analogs having at least one commonepitope with hCTLA-4. The complete hCTLA-4 sequence can be found underGenBank Accession No. AAB59385.

A “disease” refers to any disorder of structure or function in anorganism, for example a human that is not the direct result of aphysical injury. An “infectious disease” is a disease that is caused byan organism such as a bacterium, fungus, parasite virus or otherpathogen.

“Dosing interval,” as used herein, means the amount of time that elapsesbetween multiple doses of a formulation disclosed herein beingadministered to a subject. Dosing interval can thus be indicated asranges.

The term “dosing frequency” as used herein refers to the frequency ofadministering doses of a formulation disclosed herein in a given time.Dosing frequency can be indicated as the number of doses per a giventime, e.g., once a week or once in two weeks.

The use of the term “fixed dose” with regard to a composition of theinvention means that two or more different antibodies in a singlecomposition are present in the composition in particular (fixed) ratioswith each other. In some embodiments, the fixed dose is based on theweight (e.g., mg) of the antibodies. In certain embodiments, the fixeddose is based on the concentration (e.g., mg/ml) of the antibodies. Insome embodiments, the ratio is at least about 1:1, about 1:2, about 1:3,about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about1:10, about 1:15, about 1:20, about 1:30, about 1:40, about 1:50, about1:60, about 1:70, about 1:80, about 1:90, about 1:100, about 1:120,about 1:140, about 1:160, about 1:180, about 1:200, about 200:1, about180:1, about 160:1, about 140:1, about 120:1, about 100:1, about 90:1,about 80:1, about 70:1, about 60:1, about 50:1, about 40:1, about 30:1,about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1,about 6:1, about 5:1, about 4:1, about 3:1, or about 2:1 mg firstantibody to mg second antibody. For example, the 3:1 ratio of a firstantibody and a second antibody can mean that a vial can contain about240 mg of the first antibody and 80 mg of the second antibody or about 3mg/ml of the first antibody and 1 mg/ml of the second antibody.

The use of the term “flat dose” with regard to the composition of theinvention means a dose that is administered to a patient without regardfor the weight or body surface area (BSA) of the patient. The flat doseis therefore not provided as a mg/kg dose, but rather as an absoluteamount of the agent (e.g., the anti-CTLA4 antibody and/or anti-PD-1antibody). For example, a 60 kg person and a 100 kg person would receivethe same dose of the composition (e.g., 240 mg of an anti-PD-1 antibodyand 80 mg of an anti-CTLA4 antibody in a single fixed dosing formulationvial containing both 240 mg of an anti-PD-1 antibody and 80 mg of ananti-CTLA4 antibody (or two fixed dosing formulation vials containing120 mg of an anti-PD-1 antibody and 40 mg of an anti-CTLA4 antibody,etc)).

The term “weight based dose” as referred to herein means that a dosethat is administered to a patient is calculated based on the weight ofthe patient. For example, when a patient with 60 kg body weight requires3 mg/kg of an anti-PD-1 antibody in combination with 1 mg/kg of ananti-CTLA4 antibody, one can draw the appropriate amounts of theanti-PD-1 antibody (i.e., 180 mg) and the anti-CTLA4 antibody (i.e., 60mg) at once from a 3:1 ratio fixed dosing formulation of an anti-PD-1antibody and an anti-CTLA4 antibody.

The term “reference composition” as used herein refers to a compositioncomprising either the first antibody or the second antibody, but notboth. The reference composition can comprise the same components of thecomposition comprising the first antibody and the second antibody exceptthe presence of one antibody. In other embodiments, the referencecomposition is a commercially available, corresponding composition,e.g., OPDIVO® or KEYRUDA® for anti-PD-1 antibody or YERVOY® foranti-CTLA-4 antibody.

The term “GITR”, “tumor necrosis factor receptor superfamily member 18”,“activation-inducible TNFR family receptor” or “glucocorticoid-inducedTNFR-related protein” all refer to a protein that is a member of thetumor necrosis factor receptor super family. GITR is encoded for by theTNFRSF18 gene in humans. It is a 241 amino acid type I transmembraneprotein characterized by three cysteine pseudo-repeats in theextracellular domain and specifically protects T-cell receptor-inducedapoptosis, although it does not protect cells from other apoptoticsignals, including Fas triggering, dexamethasone treatment, or UVirradiation (Nocentini, G, et al. (1997) Proc. Natl. Acad. Sci, USA94:6216-622). The term GITR as used herein includes human GITR (hGITR),variants, isoforms, and species homologs of hGITR, and analogs having atleast one common epitope with hGITR. Three isoforms of hGITR have beenidentified, all of which share the same extracellular domain, except forits C-terminal portion. Variant 1 (Accession No. NP_004186) consists of241 amino acids and represents the longest transcript. It contains anextra coding segment that leads to a frame shift, compared to variant 2.The resulting protein (isoform 1) contains a distinct and shorterC-terminus, as compared to isoform 2. Variant 2 (Accession No.NP_683699) encodes the longest protein (isoform 2), consisting of 255amino acids, and is soluble. Variant 3 (Accession No. NP_683700)contains an extra coding segment that leads to a frame shift, comparedto variant 2. The resulting protein (isoform 3) contains a distinct andshorter C-terminus, as compared to isoform 2, and consists of 234 aminoacids.

The term “immunotherapy” refers to the treatment of a subject afflictedwith, or at risk of contracting or suffering a recurrence of, a diseaseby a method comprising inducing, enhancing, suppressing or otherwisemodifying an immune response.

The term “LAG3”, “LAG-3” or “Lymphocyte Activation Gene-3” refers toLymphocyte Activation Gene-3. The term LAG-3 as used herein includeshuman LAG-3 (hLAG-3), variants, isoforms, and species homologs ofhLAG-3, and analogs having at least one common epitope with hLAG-3. Theterm “human LAG-3” refers to human sequence LAG-3, such as the completeamino acid sequence of human LAG-3 having Genbank Accession No. NP002277. The term “mouse LAG-3” refers to mouse sequence LAG-3, such asthe complete amino acid sequence of mouse LAG-3 having Genbank AccessionNo. NP_032505. LAG-3 is also known in the art as, for example, CD223.The human LAG-3 sequence may differ from human LAG-3 of GenbankAccession No. NP_002277 by having, e.g., conserved mutations ormutations in non-conserved regions and the LAG-3 has substantially thesame biological function as the human LAG-3 of Genbank Accession No.NP_002277. For example, a biological function of human LAG-3 is havingan epitope in the extracellular domain of LAG-3 that is specificallybound by an antibody of the instant disclosure or a biological functionof human LAG-3 is binding to MEW Class II molecules.

The term “lyophilisate” as used herein in connection with theformulation according to the invention denotes a formulation which ismanufactured by freeze-drying methods known in the art per se. Thesolvent (e.g., water) is removed by freezing following sublimation undervacuum and desorption of residual water at elevated temperature. In thepharmaceutical field, the lyophilisate has usually residual moisture ofabout 0.1 to 5% (w/w) and is present as a powder or a physical stablecake. The lyophilisate is characterized by a fast dissolution afteraddition of a reconstitution medium.

The term “Killer Ig-like Receptor”, “Killer Inhibitory Receptor”, or“KIR”, refers to a protein or polypeptide encoded by a gene that is amember of the KIR gene family or by a cDNA prepared from such a gene. Adetailed review of the KIR gene family, including the nomenclature ofKIR genes and KIR gene products, and Genbank accession numbers forexemplary KIRs, is “The KIR Gene Cluster” by M. Carrington and P.Norman, available at the NCBI web-site called Bookshelf (accessible atncbi.nlm.nih.gov/books). The term KIR as used herein includes human KIR(hKIR), variants, isoforms, and species homologs of hKIR, and analogshaving at least one common epitope with hKIR. The sequences of human KIRgenes and cDNAs, as well as their protein products, are available inpublic databases, including GenBank. Non-limiting exemplary GenBankentries of human KIRs have the following accession numbers: KIR2DL1:Genbank accession number U24076, NM_014218, AAR16197, or L41267;KIR2DL2: Genbank accession number U24075 or L76669; KIR2DL3: Genbankaccession number U24074 or L41268; KIR2DL4: Genbank accession numberX97229; KIR2DS1: Genbank accession number X89892; KIR2DS2: Genbankaccession number L76667; KIR2DS3: Genbank accession number NM_012312 orL76670 (splice variant); KIR3DL1: Genbank accession number L41269; andKIR2DS4: Genbank accession number AAR26325. A KIR may comprise from 1 to3 extracellular domains, and may have a long (i.e., more than 40 aminoacids) or short (i.e., less than 40 amino acids) cytoplasmic tail. Aspreviously described herein, these features determine the nomenclatureof a KIR. KIR is further described in Int'l Publ. No. WO/2014/055648,which is incorporated herein by reference in its entirety.

“Programmed Death-1 (PD-1)” refers to an immunoinhibitory receptorbelonging to the CD28 family. PD-1 is expressed predominantly onpreviously activated T cells in vivo, and binds to two ligands, PD-L1and PD-L2. The term “PD-1” as used herein includes human PD-1 (hPD-1),variants, isoforms, and species homologs of hPD-1, and analogs having atleast one common epitope with hPD-1. The complete hPD-1 sequence can befound under GenBank Accession No. U64863. “PD-1” and “PD-1 receptor” areused interchangeably herein.

“Programmed Death Ligand-1 (PD-L1)” is one of two cell surfaceglycoprotein ligands for PD-1 (the other being PD-L2) that down-regulateT cell activation and cytokine secretion upon binding to PD-1. The term“PD-L1” as used herein includes human PD-L1 (hPD-L1), variants,isoforms, and species homologs of hPD-L1, and analogs having at leastone common epitope with hPD-L1. The complete hPD-L1 sequence can befound under GenBank Accession No. Q9NZQ7.

The term “reconstituted formulation” as used herein denotes aformulation which is lyophilized and re-dissolved by addition of adiluent. The diluent can contain, for example, 0.9% Sodium ChlorideInjection, USP or 5% Dextrose Injection, USP.

A “subject” includes any human or nonhuman animal. The term “nonhumananimal” includes, but is not limited to, vertebrates such as nonhumanprimates, sheep, dogs, and rodents such as mice, rats and guinea pigs.In some embodiments, the subject is a human. The terms, “subject” and“patient” are used interchangeably herein.

A “therapeutically effective amount” or “therapeutically effectivedosage” of a drug or therapeutic agent is any amount of the drug that,when used alone or in combination with another therapeutic agent,protects a subject against the onset of a disease or promotes diseaseregression evidenced by a decrease in severity of disease symptoms, anincrease in frequency and duration of disease symptom-free periods, or aprevention of impairment or disability due to the disease affliction.The ability of a therapeutic agent to promote disease regression can beevaluated using a variety of methods known to the skilled practitioner,such as in human subjects during clinical trials, in animal modelsystems predictive of efficacy in humans, or by assaying the activity ofthe agent in in vitro assays.

As used herein, “subtherapeutic dose” means a dose of a therapeuticcompound (e.g., an antibody) that is lower than the usual or typicaldose of the therapeutic compound when administered alone for thetreatment of a hyperproliferative disease (e.g., cancer).

“Treatment” or “therapy” of a subject refers to any type of interventionor process performed on, or the administration of an active agent to,the subject with the objective of reversing, alleviating, ameliorating,inhibiting, slowing down or preventing the onset, progression,development, severity or recurrence of a symptom, complication orcondition, or biochemical indicia associated with a disease.

The use of the alternative (e.g., “or”) should be understood to meaneither one, both, or any combination thereof of the alternatives. Asused herein, the indefinite articles “a” or “an” should be understood torefer to “one or more” of any recited or enumerated component.

The terms “about” or “comprising essentially of” refer to a value orcomposition that is within an acceptable error range for the particularvalue or composition as determined by one of ordinary skill in the art,which will depend in part on how the value or composition is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” or “comprising essentially of” can mean within 1 ormore than 1 standard deviation per the practice in the art.Alternatively, “about” or “comprising essentially of” can mean a rangeof up to 10% or 20% (i.e., ±10% or ±20%). For example, about 3 mg caninclude any number between 2.7 mg and 3.3 mg (for 10%) or between 2.4 mgand 3.6 mg (for 20%). Furthermore, particularly with respect tobiological systems or processes, the terms can mean up to an order ofmagnitude or up to 5-fold of a value. When particular values orcompositions are provided in the application and claims, unlessotherwise stated, the meaning of “about” or “comprising essentially of”should be assumed to be within an acceptable error range for thatparticular value or composition.

The terms “once about every week,” “once about every two weeks,” or anyother similar dosing interval terms as used herein mean approximatenumbers. “Once about every week” can include every seven days±one day,i.e., every six days to every eight days. “Once about every two weeks”can include every fourteen days±three days, i.e., every eleven days toevery seventeen days. Similar approximations apply, for example, to onceabout every three weeks, once about every four weeks, once about everyfive weeks, once about every six weeks and once about every twelveweeks. In some embodiments, a dosing interval of once about every sixweeks or once about every twelve weeks means that the first dose can beadministered any day in the first week, and then the next dose can beadministered any day in the sixth or twelfth week, respectively. Inother embodiments, a dosing interval of once about every six weeks oronce about every twelve weeks means that the first dose is administeredon a particular day of the first week (e.g., Monday) and then the nextdose is administered on the same day of the sixth or twelfth weeks(i.e., Monday), respectively.

As described herein, any concentration range, percentage range, ratiorange or integer range is to be understood to include the value of anyinteger within the recited range and, when appropriate, fractionsthereof (such as one tenth and one hundredth of an integer), unlessotherwise indicated.

Various aspects of the invention are described in further detail in thefollowing subsections.

Anti-PD-1 and Anti-PD-L1 Antibodies

The composition of the invention includes a first antibody and a secondantibody at a ratio between 1:100 to 100:1. In one aspect, the firstantibody is an anti-PD-1 antibody or an anti-PD-L1 antibody. PD-1 is akey immune checkpoint receptor expressed by activated T and B cells andmediates immunosuppression. PD-1 is a member of the CD28 family ofreceptors, which includes CD28, CTLA-4, ICOS, PD-1, and BTLA. Two cellsurface glycoprotein ligands for PD-1 have been identified, ProgrammedDeath Ligand-1 (PD-L1) and Programmed Death Ligand-2 (PD-L2), that areexpressed on antigen-presenting cells as well as many human cancers andhave been shown to down regulate T cell activation and cytokinesecretion upon binding to PD-1. Inhibition of the PD-1/PD-L1 interactionmediates potent antitumor activity in preclinical models.

HuMAbs that bind specifically to PD-1 with high affinity have beendisclosed in U.S. Pat. Nos. 8,008,449 and 8,779,105. Other anti-PD-1mAbs have been described in, for example, U.S. Pat. Nos. 6,808,710,7,488,802, 8,168,757 and 8,354,509, and PCT Publication No. WO2012/145493. Each of the anti-PD-1 HuMAbs disclosed in U.S. Pat. No.8,008,449 has been demonstrated to exhibit one or more of the followingcharacteristics: (a) binds to human PD-1 with a K_(D) of 1×10⁻⁷M orless, as determined by surface plasmon resonance using a Biacorebiosensor system; (b) does not substantially bind to human CD28, CTLA-4or ICOS; (c) increases T-cell proliferation in a Mixed LymphocyteReaction (MLR) assay; (d) increases interferon-γ production in an MLRassay; (e) increases IL-2 secretion in an MLR assay; (f) binds to humanPD-1 and cynomolgus monkey PD-1; (g) inhibits the binding of PD-L1and/or PD-L2 to PD-1; (h) stimulates antigen-specific memory responses;(i) stimulates Ab responses; and (j) inhibits tumor cell growth in vivo.Anti-PD-1 antibodies useful for the present invention include mAbs thatbind specifically to human PD-1 and exhibit at least one, preferably atleast five, of the preceding characteristics.

In one embodiment, the anti-PD-1 antibody is nivolumab. Nivolumab (alsoknown as “OPDIVO®”; formerly designated 5C4, BMS-936558, MDX-1106, orONO-4538) is a fully human IgG4 (S228P) PD-1 immune checkpoint inhibitorantibody that selectively prevents interaction with PD-1 ligands (PD-L1and PD-L2), thereby blocking the down-regulation of antitumor T-cellfunctions (U.S. Pat. No. 8,008,449; Wang et al., 2014 Cancer ImmunolRes. 2(9):846-56). In another embodiment, the anti-PD-1 antibody orfragment thereof cross-competes with nivolumab. In other embodiments,the anti-PD-1 antibody or fragment thereof binds to the same epitope asnivolumab. In certain embodiments, the anti-PD-1 antibody has the sameCDRs as nivolumab.

In another embodiment, the anti-PD-1 antibody or fragment thereofcross-competes with pembrolizumab. In some embodiments, the anti-PD-1antibody or fragment thereof binds to the same epitope as pembrolizumab.In certain embodiments, the anti-PD-1 antibody has the same CDRs aspembrolizumab. In another embodiment, the anti-PD-1 antibody ispembrolizumab. Pembrolizumab (also known as KEYTRUDA®, lambrolizumab,and MK-3475) is a humanized monoclonal IgG4 antibody directed againsthuman cell surface receptor PD-1 (programmed death-1 or programmed celldeath-1). Pembrolizumab is described, for example, in U.S. Pat. Nos.8,354,509 and 8,900,587; see alsohttp://www.cancer.gov/drugdictionary?cdrid=695789 (last accessed: Dec.14, 2014). Pembrolizumab has been approved by the FDA for the treatmentof relapsed or refractory melanoma.

In other embodiments, the anti-PD-1 antibody or fragment thereofcross-competes with MEDI0608. In still other embodiments, the anti-PD-1antibody or fragment thereof binds to the same epitope as MEDI0608. Incertain embodiments, the anti-PD-1 antibody has the same CDRs asMEDI0608. In other embodiments, the anti-PD-1 antibody is MEDI0608(formerly AMP-514), which is a monoclonal antibody. MEDI0608 isdescribed, for example, in U.S. Pat. No. 8,609,089B2 or inhttp://www.cancer.gov/drugdictionary?cdrid=756047 (last accessed Dec.14, 2014).

In certain embodiments, the first antibody is an anti-PD-1 antagonist.One example of the anti-PD-1 antagonist is AMP-224, which is a B7-DC Fcfusion protein. AMP-224 is discussed in U.S. Publ. No. 2013/0017199 orinhttp://www.cancer.gov/publications/dictionaries/cancer-drug?cdrid=700595(last accessed Jul. 8, 2015).

In other embodiments, the anti-PD-1 antibody or fragment thereofcross-competes with BGB-A317. In some embodiments, the anti-PD-1antibody or fragment thereof binds the same epitope as BGB-A317. Incertain embodiments, the anti-PD-1 antibody has the same CDRs asBGB-A317. In certain embodiments, the anti-PD-1 antibody is BGB-A317,which is a humanized monoclonal antibody. BGB-A317 is described in U.S.Publ. No. 2015/0079109.

Anti-PD-1 antibodies useful for the disclosed compositions also includeisolated antibodies that bind specifically to human PD-1 andcross-compete for binding to human PD-1 with nivolumab (see, e.g., U.S.Pat. Nos. 8,008,449 and 8,779,105; WO 2013/173223). The ability ofantibodies to cross-compete for binding to an antigen indicates thatthese antibodies bind to the same epitope region of the antigen andsterically hinder the binding of other cross-competing antibodies tothat particular epitope region. These cross-competing antibodies areexpected to have functional properties very similar to those ofnivolumab by virtue of their binding to the same epitope region of PD-1.Cross-competing antibodies can be readily identified based on theirability to cross-compete with nivolumab in standard PD-1 binding assayssuch as Biacore analysis, ELISA assays or flow cytometry (see, e.g., WO2013/173223).

In certain embodiments, the antibodies that cross-compete for binding tohuman PD-1 with, or bind to the same epitope region of human PD-1 as,nivolumab are mAbs. For administration to human subjects, thesecross-competing antibodies can be chimeric antibodies, or humanized orhuman antibodies. Such chimeric, humanized or human mAbs can be preparedand isolated by methods well known in the art.

Anti-PD-1 antibodies useful for the compositions of the disclosedinvention also include antigen-binding portions of the above antibodies.It has been amply demonstrated that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L) and C_(H1)domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the V_(H) and C_(H1) domains; and (iv) a Fvfragment consisting of the V_(L) and V_(H) domains of a single arm of anantibody.

Anti-PD-1 antibodies suitable for use in the disclosed compositions areantibodies that bind to PD-1 with high specificity and affinity, blockthe binding of PD-L1 and or PD-L2, and inhibit the immunosuppressiveeffect of the PD-1 signaling pathway. In any of the compositions ormethods disclosed herein, an anti-PD-1 “antibody” includes anantigen-binding portion or fragment that binds to the PD-1 receptor andexhibits the functional properties similar to those of whole antibodiesin inhibiting ligand binding and upregulating the immune system. Incertain embodiments, the anti-PD-1 antibody or antigen-binding portionthereof cross-competes with nivolumab for binding to human PD-1. Inother embodiments, the anti-PD-1 antibody or antigen-binding portionthereof is a chimeric, humanized or human monoclonal antibody or aportion thereof. In certain embodiments, the antibody is a humanizedantibody. In other embodiments, the antibody is a human antibody.Antibodies of an IgG1, IgG2, IgG3 or IgG4 isotype can be used.

In certain embodiments, the anti-PD-1 antibody or antigen-bindingportion thereof comprises a heavy chain constant region which is of ahuman IgG1 or IgG4 isotype. In certain other embodiments, the sequenceof the IgG4 heavy chain constant region of the anti-PD-1 antibody orantigen-binding portion thereof contains an S228P mutation whichreplaces a serine residue in the hinge region with the proline residuenormally found at the corresponding position in IgG1 isotype antibodies.This mutation, which is present in nivolumab, prevents Fab arm exchangewith endogenous IgG4 antibodies, while retaining the low affinity foractivating Fc receptors associated with wild-type IgG4 antibodies (Wanget al., 2014). In yet other embodiments, the antibody comprises a lightchain constant region which is a human kappa or lambda constant region.In other embodiments, the anti-PD-1 antibody or antigen-binding portionthereof is a mAb or an antigen-binding portion thereof. In certainembodiments of any of the therapeutic methods described hereincomprising administration of an anti-PD-1 antibody, the anti-PD-1antibody is nivolumab. In other embodiments, the anti-PD-1 antibody ispembrolizumab. In other embodiments, the anti-PD-1 antibody is chosenfrom the human antibodies 17D8, 2D3, 4H1, 4A11, 7D3 and 5F4 described inU.S. Pat. No. 8,008,449. In still other embodiments, the anti-PD-1antibody is MEDI0608 (formerly AMP-514), AMP-224, or Pidilizumab(CT-011).

In certain embodiments, the first antibody for the disclosed compositionis an anti-PD-L1 antibody. Because anti-PD-1 and anti-PD-L1 target thesame signaling pathway and have been shown in clinical trials to exhibitsimilar levels of efficacy in a variety of cancers, an anti-PD-L1antibody can be substituted for the anti-PD-1 antibody in any of thetherapeutic methods or compositions disclosed herein. In certainembodiments, the anti-PD-L1 antibody is BMS-936559 (formerly 12A4 orMDX-1105) (see, e.g., U.S. Pat. No. 7,943,743; WO 2013/173223). In otherembodiments, the anti-PD-L1 antibody is MPDL3280A (also known as RG7446and atezolizumab) (see, e.g., Herbst et al. 2013 J Clin Oncol31(suppl):3000; U.S. Pat. No. 8,217,149), MEDI4736 (Khleif, 2013, In:Proceedings from the European Cancer Congress 2013; Sep. 27-Oct. 1,2013; Amsterdam, The Netherlands. Abstract 802) or MSB0010718C (alsocalled Avelumab; See US 2014/0341917). In certain embodiments, theantibodies that cross-compete for binding to human PD-L1 with, or bindto the same epitope region of human PD-L1 as the above-references PD-L1antibodies are mAbs. For administration to human subjects, thesecross-competing antibodies can be chimeric antibodies, or can behumanized or human antibodies. Such chimeric, humanized or human mAbscan be prepared and isolated by methods well known in the art.

Anti-CTLA-4 Antibodies

Anti-CTLA-4 antibodies used for the instant invention bind to humanCTLA-4 so as to disrupt the interaction of CTLA-4 with a human B7receptor. Because the interaction of CTLA-4 with B7 transduces a signalleading to inactivation of T-cells bearing the CTLA-4 receptor,disruption of the interaction effectively induces, enhances or prolongsthe activation of such T cells, thereby inducing, enhancing orprolonging an immune response.

HuMAbs that bind specifically to CTLA-4 with high affinity have beendisclosed in U.S. Pat. Nos. 6,984,720 and 7,605,238. Other anti-CTLA-4mAbs have been described in, for example, U.S. Pat. Nos. 5,977,318,6,051,227, 6,682,736, and 7,034,121. The anti-CTLA-4 HuMAbs disclosed inU.S. Pat. Nos. 6,984,720 and 7,605,238 have been demonstrated to exhibitone or more of the following characteristics: (a) binds specifically tohuman CTLA-4 with a binding affinity reflected by an equilibriumassociation constant (K_(a)) of at least about 10⁷M⁻¹, or about 10⁹M⁻¹,or about 10¹⁰ M⁻¹ to 10¹¹ M⁻¹ or higher, as determined by Biacoreanalysis; (b) a kinetic association constant (k_(a)) of at least about10³, about 10⁴, or about 10⁵ m⁻¹ s⁻¹; (c) a kinetic disassociationconstant (k_(d)) of at least about 10³, about 10⁴, or about 10⁵ m⁻¹ s⁻¹;and (d) inhibits the binding of CTLA-4 to B7-1 (CD80) and B7-2 (CD86).Anti-CTLA-4 antibodies useful for the present invention include mAbsthat bind specifically to human CTLA-4 and exhibit at least one, atleast two, or at least three of the preceding characteristics. Anexemplary clinical anti-CTLA-4 antibody is the human mAb 10D1 (now knownas ipilimumab and marketed as YERVOY®) as disclosed in U.S. Pat. No.6,984,720.

An exemplary clinical anti-CTLA-4 antibody is the human mAb 10D1 (nowknown as ipilimumab and marketed as YERVOY®) as disclosed in U.S. Pat.No. 6,984,720. Ipilimumab is an anti-CTLA-4 antibody for use in themethods disclosed herein. Ipilimumab is a fully human, IgG1 monoclonalantibody that blocks the binding of CTLA-4 to its B7 ligands, therebystimulating T cell activation and improving overall survival (OS) inpatients with advanced melanoma.

Another anti-CTLA-4 antibody useful for the present methods istremelimumab (also known as CP-675,206). Tremelimumab is human IgG2monoclonal anti-CTLA-4 antibody. Tremelimumab is described inWO/2012/122444, U.S. Publ. No. 2012/263677, or WO Publ. No. 2007/113648A2.

Anti-CTLA-4 antibodies useful for the disclosed composition also includeisolated antibodies that bind specifically to human CTLA-4 andcross-compete for binding to human CTLA-4 with ipilimumab ortremelimumab or bind to the same epitope region of human CTLA-4 asipilimumab or tremelimumab. In certain embodiments, the antibodies thatcross-compete for binding to human CTLA-4 with, or bind to the sameepitope region of human CTLA-4 as does ipilimumab or tremelimumab, areantibodies comprising a heavy chain of the human IgG1 isotype. Foradministration to human subjects, these cross-competing antibodies arechimeric antibodies, or humanized or human antibodies. Usefulanti-CTLA-4 antibodies also include antigen-binding portions of theabove antibodies such as Fab, F(ab′)₂, Fd or Fv fragments.

Anti-LAG-3 Antibodies

Anti-LAG-3 antibodies of the instant invention bind to human LAG-3.Antibodies that bind to LAG-3 have been disclosed in Int'l Publ. No.WO/2015/042246 and U.S. Publ. Nos. 2014/0093511 and 2011/0150892.

An exemplary LAG-3 antibodies useful for the present invention is 25F7(described in U.S. Publ. No. 2011/0150892). An additional exemplaryLAG-3 antibody useful for the present invention is BMS-986016. In oneembodiment, an anti-LAG-3 antibody useful for the compositioncross-competes with 25F7 or BMS-986016. In another embodiment, ananti-LAG-3 antibody useful for the composition binds to the same epitopeas 25F7 or BMS-986016. In other embodiments, an anti-LAG-3 antibodycomprises six CDRs of 25F7 or BMS-986016.

Anti-CD137 Antibodies

Anti-CD137 antibodies specifically bind to and activate CD137-expressingimmune cells, stimulating an immune response, in particular a cytotoxicT cell response, against tumor cells. Antibodies that bind to CD137 havebeen disclosed in U.S. Publ. No. 2005/0095244 and U.S. Pat. Nos.7,288,638, 6,887,673, 7,214,493, 6,303,121, 6,569,997, 6,905,685,6,355,476, 6,362,325, 6,974,863, and 6,210,669.

In some embodiments, the anti-CD137 antibody is urelumab (BMS-663513),described in U.S. Pat. No. 7,288,638 (20H4.9-IgG4 [1007 or BMS-663513]).In some embodiments, the anti-CD137 antibody is BMS-663031(20H4.9-IgG1), described in U.S. Pat. No. 7,288,638. In someembodiments, the anti-CD137 antibody is 4E9 or BMS-554271, described inU.S. Pat. No. 6,887,673. In some embodiments, the anti-CD137 antibody isan antibody disclosed in U.S. Pat. Nos. 7,214,493; 6,303,121; 6,569,997;6,905,685; or 6,355,476. In some embodiments, the anti-CD137 antibody is1D8 or BMS-469492; 3H3 or BMS-469497; or 3E1, described in U.S. Pat. No.6,362,325. In some embodiments, the anti-CD137 antibody is an antibodydisclosed in issued U.S. Pat. No. 6,974,863 (such as 53A2). In someembodiments, the anti-CD137 antibody is an antibody disclosed in issuedU.S. Pat. No. 6,210,669 (such as 1D8, 3B8, or 3E1). In some embodiments,the antibody is Pfizer's PF-05082566 (PF-2566) In other embodiments, ananti-CD137 antibody useful for the invention cross-competes with theanti-CD137 antibodies disclosed herein. In some embodiments, ananti-CD137 antibody binds to the same epitope as the anti-CD137 antibodydisclosed herein. In other embodiments, an anti-CD137 antibody usefulfor the invention comprises six CDRs of the anti-CD137 antibodiesdisclosed herein.

Anti-KIR Antibodies

Antibodies that bind specifically to KIR block interaction betweenKiller-cell immunoglobulin-like receptors (KIR) on NK cells with theirligands. Blocking these receptors facilitates activation of NK cellsand, potentially, destruction of tumor cells by the latter. Examples ofanti-KIR antibodies have been disclosed in Int'l Publ. Nos.WO/2014/055648, WO 2005/003168, WO 2005/009465, WO 2006/072625, WO2006/072626, WO 2007/042573, WO 2008/084106, WO 2010/065939, WO2012/071411 and WO/2012/160448.

One anti-KIR antibody useful for the present invention is lirilumab(also referred to as BMS-986015, IPH2102, or the S241P variant of1-7F9), first described in Int'l Publ. No. WO 2008/084106. An additionalanti-KIR antibody useful for the present invention is 1-7F9 (alsoreferred to as IPH2101), described in Int'l Publ. No. WO 2006/003179. Inone embodiment, an anti-KIR antibody for the present composition crosscompetes for binding to KIR with lirilumab or I-7F9. In anotherembodiment, an anti-KIR antibody binds to the same epitope as lirilumabor I-7F9. In other embodiments, an anti-KIR antibody comprises six CDRsof lirilumab or I-7F9.

Anti-GITR Antibodies

Anti-GITR antibodies for combining with an anti-PD-1 antibody in a fixeddose may be any anti-GITR antibody that binds specifically to human GITRtarget and activate the glucocorticoid-induced tumor necrosis factorreceptor (GITR). GITR is a member of the TNF receptor superfamily thatis expressed on the surface of multiple types of immune cells, includingregulatory T cells, effector T cells, B cells, natural killer (NK)cells, and activated dendritic cells (“anti-GITR agonist antibodies”).Specifically, GITR activation increases the proliferation and functionof effector T cells, as well as abrogating the suppression induced byactivated T regulatory cells. In addition, GITR stimulation promotesanti-tumor immunity by increasing the activity of other immune cellssuch as NK cells, antigen presenting cells, and B cells. Examples ofanti-GITR antibodies have been disclosed in Int'l Publ. Nos.WO/2015/031667, WO2015/184,099, WO2015/026,684, WO11/028683 andWO/2006/105021, U.S. Pat. Nos. 7,812,135 and 8,388,967 and U.S. Publ.Nos. 2009/0136494, 2014/0220002, 2013/0183321 and 2014/0348841.

In one embodiment, an anti-GITR antibody useful for the presentinvention is TRX518 (described in, for example, Schaer et al. Curr OpinImmunol. (2012) April; 24(2): 217-224, and WO/2006/105021). In anotherembodiment, an anti-GITR antibody useful for the present invention isMK4166 or MK1248 and antibodies described in WO11/028683 and in U.S.Pat. No. 8,709,424, and comprising, e.g., a VH chain comprising SEQ IDNO: 104 and a VL chain comprising SEQ ID NO: 105, wherein the SEQ ID NOsare from WO11/028683 or U.S. Pat. No. 8,709,424). In certainembodiments, an anti-GITR antibody is an anti-GITR antibody that isdisclosed in WO2015/031667, e.g., an antibody comprising VH CDRs 1-3comprising SEQ ID NOs: 31, 71 and 63 of WO2015/031667, respectively, andVL CDRs 1-3 comprising SEQ ID NOs: 5, 14 and 30 of WO2015/031667. Incertain embodiments, an anti-GITR antibody is an anti-GITR antibody thatis disclosed in WO2015/184099, e.g., antibody Hum231#1 or Hum231#2, orthe CDRs thereof, or a derivative thereof (e.g., pab1967, pab1975 orpab1979). In certain embodiments, an anti-GITR antibody is an anti-GITRantibody that is disclosed in JP2008278814, WO09/009116, WO2013/039954,US20140072566, US20140072565, US20140065152, or WO2015/026684, or isINBRX-110 (INHIBRx), LKZ-145 (Novartis), or MEDI-1873 (MedImmune). Incertain embodiments, an anti-GITR antibody is an anti-GITR antibody thatis described in PCT/US2015/033991 (e.g., an antibody comprising thevariable regions of 28F3, 18E10 or 19D3). For example, an anti-GITRantibody may be an antibody comprising the following VH and VL chains orthe CDRs thereof:

VH: (SEQ ID NO: 1) QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYEGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSMVRGDYYYGMDVWGQGTTVTVS, and VL: (SEQ ID NO: 2)AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPYTFGQ GTKLEIK; or VH:(SEQ ID NO: 3) QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGFHWVRQAPGKGLEWVAVIWYAGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGQLDYYYYYVMDVWGQGTTVTVSS, and VL: (SEQ ID NO: 4)DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQ GTKLEIK; or VH:(SEQ ID NO: 5) VQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYAGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGRIAVAFYYSMDVWGQGTTVTVSS, and VL: (SEQ ID NO: 6)DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQ GTKLEIK.

In certain embodiments, an antibody comprising a pair of the above VHand VL light chains, or their CDRs, comprises a heavy chain constantregion of an IgG1 isotype, either wild type or mutated, e.g., to beeffectorless. In one embodiment, an anti-GITR antibody comprises thefollowing heavy and light chains amino acid sequences:

heavy chain: (SEQ ID NO: 7)QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYEGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSMVRGDYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, and light chain:(SEQ ID NO: 8) AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC, orheavy chain: (SEQ ID NO: 9)QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYEGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSMVRGDYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG, and light chain:(SEQ ID NO: 8) AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC.

In certain embodiments, an anti-GITR antibody for the presentcomposition cross-competes with an anti-GITR antibody described herein,e.g., TRX518, MK4166 or an antibody comprising a VH domain and a VLdomain amino acid sequence described herein. In some embodiments, ananti-GITR antibody for the present composition binds the same epitope asthat of an anti-GITR antibody described herein, e.g., TRX518, MK4166 oran antibody comprising a VH domain and a VL domain amino acid sequencedescribed herein. In certain embodiments, an anti-GITR antibodycomprises the six CDRs of TRX518, MK4166 or those of an antibodycomprising a VH domain and a VL domain amino acid sequence describedherein. An exemplary pharmaceutical composition comprises an anti-PD-1antibody, e.g., nivolumab, MK-3475 (pembrolizumab) or atezolizumab, andan anti-GITR agonist antibody, e.g., TRX518, MK4166 or an antibodycomprising a VH domain and a VL domain amino acid sequence describedherein, wherein the ratio of the amount (e.g., concentration (e.g.,mg/ml) or weight (e.g., mg)) of the anti-PD-1 antibody to the amount ofthe anti-GITR antibody (e.g., concentration (e.g., mg/ml) or weight(e.g., mg), respectively) is from about 1:1-20; about 1:1-10; about1:1-5; about 1:2-5; about 1:2-3; about 1:3-5; about 1-20:1; about1-10:1; about 1-5:1; about 2-5:1; about 2-3:1; or about 3-5:1. Forexample, the ratio of (i) an anti-PD-1 or anti-PD-L1 antibody to (2) ananti-GITR antibody, may be 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2,1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1. “:” refers to “to,”e.g., “1:1-20” refers to a ratio of 1 to a number selected from 1-20.The combination may be administered weekly, biweekly, once every threeweeks or monthly.

In certain embodiments, an anti-PD-1 or anti-PD-L1 antibody e.g.,nivolumab, pembrolizumab or atezolizumab, is co-formulated with ananti-GITR antibody, wherein the anti-GITR antibody is at a dose, e.g.,flat dose, of 0.1 to 1000 mg, such as 0.1 to 100 mg, 0.5 to 100 mg, 1 to100 mg, 5 to 100 mg, 10 to 100 mg, 50 to 100 mg, 0.1 to 300 mg, 0.5 to300 mg, 1 to 300 mg, 5 to 300 mg, 10 to 300 mg, 50 to 300 mg, 100 to 300mg or 200 to 300 mg. Exemplary amounts of anti-GITR antibody that may beco-formulated with an anti-PD-1 or anti-PD-L1 antibody include about 0.1mg, about 0.3 mg, about 0.5 mg, about 1 mg, about 3 mg, about 10 mg,about 30 mg, about 100 mg, about 200 mg, about 240 mg, about 250 mg,about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg,about 800 mg, about 900 mg or about 1000 mg. In certain embodiments, ananti-PD-1 or anti-PD-L1 antibody is co-formulated with an anti-GITRantibody, wherein the dose of the anti-PD-1 or PD-L1 antibody, is a dose(e.g., flat dose) of 100-300 mg, such as, 200-300 mg, 220-260 mg,230-250 mg or 240 mg, such as about 60 mg, about 80 mg, about 100 mg,about 120 mg, about 140 mg, about 160 mg, about 180 mg, about 200 mg,about 220 mg, about 240 mg, about 260 mg, about 280 or about 300 mg.

In exemplary embodiments, an anti-PD-1 or anti-PD-L1 antibody, e.g.,nivolumab, pembrolizumab or atezolizumab, is co-formulated with ananti-GITR antibody, e.g., an antibody comprising (i) a VH and a VLdomain comprising the amino acid sequences of SEQ ID NOs: 1 and 2,respectively, SEQ ID NOs: 3 and 4, respectively, or SEQ ID NOs: 5 and 6,respectively; or the VH CDR1, CDR2, CDR3 and the VL CDR1, CDR2 and CDR3of any of these pairs of variable regions, or (ii) the heavy and lightchains comprising the amino acid sequences of SEQ ID NOs 7 and 8,respectively, or SEQ ID NOs: 7 and 9, respectively, at the followingfixed dose ratios: 80-300 mg of anti-PD-1 or anti-PD-L1 antibody to1-1000 mg of anti-GITR antibody; 80-300 mg of anti-PD-1 or anti-PD-L1antibody to 1-100 mg of anti-GITR antibody; 80-300 mg of anti-PD-1 oranti-PD-L1 antibody to 10-100 mg of anti-GITR antibody; 80-300 mg ofanti-PD-1 or anti-PD-L1 antibody to 10-300 mg of anti-GITR antibody or80-300 mg of anti-PD-1 or anti-PD-L1 antibody to 100-300 mg of anti-GITRantibody. In exemplary embodiments, nivolumab is co-formulated with ananti-GITR antibody, wherein the dose of nivolumab is about 80 mg orabout 240 mg. A fixed dose combination may be administered as anintravenous infusion over, e.g., about 30, about 30-60, about 60 orabout 60-90 minutes every about 1, about 2, about 3 or about 4 weeks.

In certain embodiments, about 3 mg/kg anti-PD-1 antibody, e.g.,nivolumab, may be administered together, e.g., as a fixed dosecombination, with about 0.1-10 mg/kg, about 0.1-5 mg/kg, about 0.5-10mg/kg, about 0.5-5 mg/kg, about 0.5-2 mg/kg, about 1-2 mg/kg or about2-5 mg/kg anti-GITR antibody, e.g., TRX518, MK4166, or an antibodycomprising the heavy and light chains or variable regions or CDRsdescribed herein, e.g., as an intravenous infusion over, e.g., about 30,about 30-60, about 60 or about 60-90 minutes every about 1, about 2,about 3 or about 4 weeks. In certain embodiments, about 2 mg/kganti-PD-1 antibody, e.g., nivolumab or MK-3475, is administeredtogether, e.g., as a fixed dose combination, with about 0.1-10 mg/kg,about 0.1-5 mg/kg, about 0.5-10 mg/kg, about 0.5-5 mg/kg, about 0.5-2mg/kg, about 1-2 mg/kg or about 2-5 mg/kg anti-GITR antibody, e.g.,MK4166 or an antibody comprising the heavy and light chains or variableregions or CDRs described herein, e.g., as an intravenous infusion over,e.g., about 30, about 30-60 or about 60 minutes every about 1, about 2,about 3 or about 4 weeks. The amount of antibodies in mg/kg can becalculated to determine the weight (mg) or the concentration (mg/ml) ofthe antibodies required for a fixed dosing ratio formulation. In certainembodiments, an anti-PD-1 antibody and an anti-GITR antibody areprovided as a lyophilized composition, e.g., in a vial or a dual chambersyringe. A lyophilized composition can comprise, e.g., about 50 mg of ananti-PD-1 or anti-PD-L1 antibody, e.g., nivolumab, MK3475 oratezolizumab and about 5-250 mg, about 10-250, about 30-100 mg, about30-70 mg or about 50 mg of an anti-GITR antibody, e.g., TRX-518, MK4166or an antibody comprising the heavy and light chains or variable regionsor CDRs described herein.

Additional Antibodies

In some embodiments, the second antibody to be combined with the firstantibody is an anti-TGFβ antibody, as disclosed in Int'l Publ. No.WO/2009/073533. In some embodiments, the second antibody is ananti-IL-10 antibody, as disclosed in Int'l Publ. No. WO/2009/073533. Insome other embodiments, the second antibody is an anti-B7-H4 antibody,as disclosed in Int'l Publ. No. WO/2009/073533. In certain embodiments,the second antibody is an anti-Fas ligand antibody, as disclosed inInt'l Publ. No. WO/2009/073533. In some embodiments, the second antibodyis an anti-CXCR4 antibody, as disclosed in U.S. Publ. No. 2014/0322208(e.g., Ulocuplumab (BMS-936564)). In some embodiments is the secondantibody is an anti-mesothelin antibody, as disclosed in U.S. Pat. No.8,399,623. In some embodiments, the second antibody is an anti-HER2antibody, for example, Herceptin (U.S. Pat. No. 5,821,337), trastuzumab,or ado-trastuzumab emtansine (Kadcyla, e.g., WO/2001/000244). Inembodiments, the second antibody to be combined with the first antibodyis an anti-CD27 antibody. In embodiments, the anti-CD-27 antibody isVarlilumab (also known as “CDX-1127” and “1F5”), which is a human IgG1antibody that is an agonist for human CD27, as disclosed in, forexample, U.S. Pat. No. 9,169,325. In some embodiments, the secondantibody to be combined with the first antibody is an anti-CD73antibody. In certain embodiments, the anti-CD73 antibody isCD73.4.IgG2C219S.IgG1.1f.

Formulations, Pharmaceutical Compositions and Dosages

In the formulation of the present invention, a first antibody and asecond antibody are formulated in a single composition of the presentinvention e.g., a pharmaceutical composition containing the firstantibody and the second antibody and a pharmaceutically acceptablecarrier. In one embodiment, the first antibody is an anti-PD-1 antibody.In another embodiment, the first antibody is an anti-PD-L1 antibody. Ananti-PD-L1 antibody can be used in place of an anti-PD-1 antibody in anycomposition or method described herein.

As used herein, a “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. In certain embodiments, the carrier for acomposition containing an antibody is suitable for intravenous,intramuscular, subcutaneous, parenteral, spinal or epidermaladministration (e.g., by injection or infusion). A pharmaceuticalcomposition of the invention can include one or more pharmaceuticallyacceptable salts, anti-oxidant, aqueous and non-aqueous carriers, and/oradjuvants such as preservatives, wetting agents, emulsifying agents anddispersing agents.

In one embodiment, the composition comprising the first antibody and thesecond antibody is provided in a single-use vial. In another embodiment,the composition comprising the first antibody and the second antibody isprovided in a multi-use vial.

In other embodiments, the first antibody (e.g., an anti-PD-1 antibody oranti-PD-L1 antibody) is formulated with any known second antibody. Insome embodiments, the second antibody is an anti-CTLA4 antibody. Incertain embodiments, the anti-CTLA4 antibody is tremelimumab oripilimumab. In some embodiments, the second antibody is an anti-CD137antibody. In some embodiments, the anti-CD137 antibody is urelumab. Insome embodiments, the second antibody is an anti-LAG3 antibody. In someembodiments, the anti-LAG3 antibody is 25F7. In some embodiments, thesecond antibody is an anti-GITR antibody. In some embodiments, theanti-GITR antibody is MK4166, TRX518, an antibody comprising the CDRs,variable chains or heavy and light chains of the anti-GITR antibodiesthat are described in PCT/US2015/033991 (e.g., those of 28F3, 18E10 or19D3) or any other anti-GITR antibody described herein. In someembodiments, the second antibody is an anti-KIR antibody. In someembodiments, the anti-KIR antibody is 1-7F9 or lirilumab. In someembodiments, the second antibody is an anti-TGFβ antibody, an anti-IL-10antibody, an anti-B7-H4 antibody, an anti-Fas ligand antibody, ananti-CXCR4 antibody, an anti-mesothelin antibody, an anti-CD27 antibody,an anti-CD73 antibody or any combination thereof.

In some embodiments, the first antibody and the second antibody arepresent in the composition at a fixed dose (i.e. a fixed ratio). Inother embodiments, this fixed dose is between at least about 1:200 to atleast about 200:1, at least about 1:150 to at least about 150:1, atleast about 1:100 to at least about 100:1, at least about 1:75 to atleast about 75:1, at least about 1:50 to at least about 50:1, at leastabout 1:25 to at least about 25:1, at least about 1:10 to at least about10:1, at least about 1:5 to at least about 5:1, at least about 1:4 to atleast about 4:1, at least about 1:3 to at least about 3:1, or at leastabout 1:2 to at least about 2:1 mg anti-PD-1 antibody (or anti-PD-L1antibody) to mg second antibody. In some embodiments, the fixed dose isat least about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about1:80, about 1:90, about 1:100, about 1:120, about 1:140, about 1:160,about 1:180, or about 1:200 anti-PD-1 antibody (or anti-PD-L1 antibody)to second antibody. In some embodiments, the fixed dose is at leastabout 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 30:1, about40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, about100:1, about 120:1, about 140:1, about 160:1, about 180:1, or about200:1 mg first antibody to mg second antibody.

In other embodiments, the composition comprises a first antibody and asecond antibody at a ratio (e.g., from 200:1 to 1:200, 100:1 to 1:100,20-1:1 to 1:1-20, or any ratio disclosed herein), wherein thecomposition has one or more characteristics selected from the groupconsisting of: (i) the aggregation in the composition is comparable tothe aggregation in a reference composition (i.e., a compositioncomprising either the first antibody or the second antibody) after6-months storage at 2° C. to 8° C.; (ii) the fragmentation in thecomposition is comparable to the aggregation in a reference composition(i.e., a composition comprising either the first antibody or the secondantibody) after 6-months storage at 2° C. to 8° C.; (iii) thedeamidation of the first antibody or the second antibody in thecomposition is comparable to the deamidation of the antibody in areference composition (i.e., a composition comprising either the firstantibody or the second antibody) after 6-months storage at 2° C. to 8°C.; (iv) the level of particulate matter in the composition iscomparable to the level of particular matter in a reference composition(i.e., a composition comprising either the first antibody or the secondantibody) after 6-months storage at 2° C. to 8° C.; and (v) anycombination thereof.

In yet other embodiments, the composition comprises a first antibody anda second antibody at a ratio (e.g., from 200:1 to 1:200, 100:1 to 1:100,20-1:1 to 1:1-20, or any ratio disclosed herein, wherein the compositionhas one or more characteristics selected from the group consisting of:(i) the aggregation in the composition is comparable to the aggregationin a reference composition (i.e., a composition comprising either thefirst antibody or the second antibody) after 6-months storage at 25° C.;(ii) the fragmentation in the composition is comparable to theaggregation in a reference composition (i.e., a composition comprisingeither the first antibody or the second antibody) after 6-months storageat 25° C.; (iii) the deamidation of the first antibody or the secondantibody in the composition are comparable to the deamidation of theantibody in a reference composition (i.e., a composition comprisingeither the first antibody or the second antibody) after 6-months storageat 25° C.; (iv) the level of particulate matter in the composition iscomparable to the level of particular matter in a reference composition(i.e., a composition comprising either the first antibody or the secondantibody) after 6-months storage at 25° C.; and (v) any combinationthereof.

In some embodiments, the aggregation of a composition is measured by alevel of high molecular weight (HMW) species in the composition, whichcan be detected by size exclusion high-performance liquid chromatography(SE-HPLC). In some embodiments, the fragmentation of a composition ismeasured by a level of low molecular weight (LMW) species in thecomposition, which is detected by SE-HPLC. In some embodiments, thedeamidation of a composition is measured by a level of acidic chargevariants in the composition, which is detected by cation exchangechromatography (CEX) or imaged capillary isoelectric focusing (iCIEF).

In some embodiments, the amount of the anti-PD-1 antibody in thecomposition is at least about 60 mg, about 80 mg, about 100 mg, about120 mg, about 140, about 160 mg, about 180 mg, about 200 mg, about 220mg, about 240 mg, about 260 mg, about 280 mg, or about 300 mg. In someembodiments, the amount of the anti-PD-1 antibody in the composition isat least about 310 mg, about 320 mg, about 330 mg, about 340 mg, about350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, or about500 mg. In some embodiments, the amount of the anti-PD-1 antibody in thecomposition is between about 60 mg and about 300 mg, between about 60 mgand about 100 mg, between about 100 mg and about 200 mg, or betweenabout 200 mg and about 300 mg. In some embodiments, the amount of theanti-PD-1 antibody in the composition is between about 300 mg and about500 mg, between about 300 mg and about 450 mg, between about 300 mg andabout 400 mg, between about 300 mg and about 350 mg, between about 350mg and about 500 mg, between about 400 mg and about 500 mg, or betweenabout 450 mg and about 500 mg. In some embodiments, the amount of theanti-PD-1 antibody in the composition is at least about 80 mg, about 160mg, or about 240 mg. In some embodiments, the amount of the anti-PD-1antibody in the composition is at least about 240 mg or at least about80 mg. In some embodiments, the amount of the anti-PD-1 antibody in thecomposition is at least about 360 mg or at least about 480 mg. In someembodiments, the amount of the anti-PD-1 antibody in the composition isa least about 0.5 mg/kg, at least about 1 mg/kg, at least about 2 mg/kg,at least about 3 mg/kg or at least about 5 mg/kg. In some embodiments,the amount of anti-PD-1 antibody in the composition is between about 0.5mg/kg and about 5 mg/kg, between about 0.5 mg/kg and about 5 mg/kg,between about 0.5 mg/kg and about 3 mg/kg or between about 0.5 mg/kg andabout 2 mg/kg. In some embodiments, the amount of the anti-PD-1 antibodyin the composition is at least about 1 mg/kg. In some embodiments, theanti-PD-1 antibody is nivolumab or pembrolizumab.

In some embodiments, the anti-PD-1 antibody is pembrolizumab and theamount of anti-PD-1 antibody in the composition is at least about 50 mg,at least about 75 mg, at least about 100 mg, at least about 150 mg, atleast about 200 mg, at least about 250 mg, or at least about 300 mg. Insome embodiments, the amount of anti-PD-1 antibody in the composition isat least about 100 mg or at least about 200 mg. In some embodiments, theamount of the anti-PD-1 antibody in the composition is at least about300 mg, at least about 350 mg, at least about 400 mg, at least about 450mg, or at least about 500 mg. In some embodiments, the anti-PD-1antibody is pembrolizumab and the amount of anti-PD-1 antibody used totreat a disease or condition can be a weight based dose, e.g., at leastabout 0.5 mg/kg, at least about 1 mg/kg, at least about 2 mg/kg, atleast about 3 mg/kg, at least about 5 mg/kg, at least about 10 mg/kg, atleast about 15 mg/kg or at least about 20 mg/kg. In some embodiments,the amount of anti-PD-1 antibody that can be used to treat a disease orcondition is a weight based dose, e.g., at least about 1 mg/kg, at leastabout 2 mg/kg, or at least about 10 mg/kg. In some embodiments, thesecond antibody is an anti-CTLA4 antibody, and the fixed dose is about1:1, about 3:1 or about 1:3 mg anti-PD-1 antibody to mg anti-CTLA4antibody. In some embodiments, the amount of the anti-CTLA4 antibody inthe composition is at least about 60 mg, about 80 mg, about 100 mg,about 120 mg, about 140, about 160 mg, about 180 mg, about 200 mg, about220 mg, about 240 mg, about 260 mg, about 280 mg, or about 300 mg. Insome embodiments, the amount of the anti-CTLA4 antibody in thecomposition is between about 60 mg and about 300 mg, between about 60 mgand about 100 mg, between about 100 mg and about 200 mg, or betweenabout 200 mg and about 300 mg. In some embodiments, the amount of theanti-CTLA4 antibody in the composition is at least about 80 mg, about160 mg, or about 240 mg. In some embodiments, the amount of theanti-CTLA4 antibody in the composition is at least about 240 mg. In someembodiments, the amount of the anti-CTLA4 antibody in the composition isat least about 1 mg/kg, at least about 2 mg/kg, at least about 3 mg/kgor at least about 5 mg/kg. In some embodiments, the amount of theanti-CTLA4 antibody in the composition is used as a weight based dose,e.g., between about 1 mg/kg and about 10 mg/kg, between about 1 mg/kgand about 5 mg/kg or between about 2 mg/kg and about 5 mg/kg. In someembodiments, the amount of the anti-CTLA4 antibody in the composition isa least about 3 mg/kg. In some embodiments, (i) the X amount is about240 mg and the Y amount is about 80 mg, (ii) the X amount is about 80 mgand the Y amount is about 80 mg; (iii) the X amount is about 160 mg andthe Y amount is about 160 mg; (iv) the X amount is about 240 mg and theY amount is about 240 mg; or (v) the X amount is about 80 mg and the Yamount is about 240 mg.

In some embodiments, the second antibody is an anti-KIR antibody and thefixed dose is about 30:1, about 10:1, about 3:1, about 1:1, about 1:2,or about 3:10 mg anti-PD-1 antibody to mg anti-KIR antibody.

In some embodiments, the second antibody is an anti-LAG3 antibody, andthe fixed dose is about 80:3, about 80:1, about 12:1, about 3:1, orabout 1:1 mg anti-PD-1 antibody to mg anti-LAG3 antibody. In someembodiments, the amount of anti-LAG3 antibody in the composition is atleast about 60 mg, about 80 mg, about 100 mg, about 120 mg, about 140,about 160 mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg,about 260 mg, about 280 mg, about 300 mg, or about 350 mg. In someembodiments, the amount of anti-LAG3 antibody in the composition isbetween about 60 and about 350 mg, between about 60 and about 300 mg,between about 100 and about 300 mg, or between about 150 and about 250mg. In some embodiments, the amount of anti-LAG3 antibody is at leastabout 240 mg.

In some embodiments the second antibody is an anti-CD137 antibody andthe fixed dose is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5,about 1:10, about 10:1, about 5:1, about 4:1 or about 2:1 mg anti-PD-1antibody to mg anti-CD137 antibody. In some embodiments, the amount ofthe anti-CD137 antibody in the composition is at least about 1 mg, atleast about 2 mg, at least about 3 mg, at least about 4 mg, at leastabout 5 mg, at least about 6 mg, at least about 7 mg, at least about 8mg, at least about 9 mg, at least about 10 mg, at least about 12 mg, atleast about 15 mg, or at least about 20 mg. In some embodiments, theamount of the anti-CD137 antibody in the composition is between about 1mg and about 20 mg, between about 1 mg and about 15 mg, between about 5mg and about 12 mg or between about 5 mg and about 10 mg. In someembodiments, the amount of the anti-CD137 antibody in the composition isa least about 8 mg.

In some embodiments the second antibody is an anti-CD73 antibody, andthe fixed dose is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5,about 1:10, about 10:1, about 5:1, about 4:1 or about 2:1 mg anti-PD-1antibody to mg anti-CD73 antibody. In some embodiments, the amount ofthe anti-CD73 antibody in the composition is from about 100 mg to about2000 mg or from about 150 mg to about 1600 mg. In some embodiments, theamount of the anti-CD73 antibody in the composition is at least about100 mg, 150 mg, 200 mg, 300 mg, 500 mg, 600 mg, 800 mg, 1000 mg, 1200mg, or 1600 mg.

In certain embodiments, the anti-CD73 antibody CD73.4.IgG2C219S.IgG1.1fand nivolumab are administered as fixed doses at one of followingcombination doses: 50 mg of anti-CD73 antibody and 240 mg of nivolumabevery two weeks; 50 mg of anti-CD73 antibody and 360 mg of nivolumabevery three weeks; 150 mg of anti-CD73 antibody and 240 mg of nivolumabevery two weeks; 150 mg of anti-CD73 antibody and 360 mg of nivolumabevery three weeks; 300 mg of anti-CD73 antibody and 240 mg of nivolumabevery two weeks; 300 mg of anti-CD73 antibody and 360 mg of nivolumabevery three weeks; 600 mg of anti-CD73 antibody and 240 mg of nivolumabevery two weeks; 600 mg of anti-CD73 antibody and 360 mg of nivolumabevery three weeks; 1200 mg of anti-CD73 antibody and 240 mg of nivolumabevery two weeks; 1200 mg of anti-CD73 antibody and 360 mg of nivolumabevery three weeks; 1600 mg of anti-CD73 antibody and 240 mg of nivolumabevery two weeks; 1600 mg of anti-CD73 antibody and 360 mg of nivolumabevery three weeks; 2000 mg of anti-CD73 antibody and 240 mg of nivolumabevery two weeks; 2000 mg of anti-CD73 antibody and 360 mg of nivolumabevery three weeks.

In some embodiments, the PD-1 antibody and the second antibody arecombined using the current formulations of the two antibodies (forexample, 2 mls of an anti-PD-1 antibody in a citrate-based buffer arecombined with 2 mls of an anti-CTLA4 antibody in a Tris-based bufferwith no buffer exchange).

In some embodiments, the composition comprises one or more additionalcomponents selected from the group consisting of: a bulking agent, astabilizing agent, a chelating agent, a surfactant, a buffering agent,and any combination thereof. In some embodiments, the buffering agentcontains a citrate buffer, a Tris buffer, a Tris-Cl buffer, a histidinebuffer, a TAE buffer, a HEPES buffer, a TBE buffer, a sodium phosphatebuffer, a MES buffer, an ammonium sulfate buffer, a potassium phosphatebuffer, a potassium thiocyanate buffer, a succinate buffer, a tartratebuffer, a DIPSO buffer, a HEPPSO buffer, a POPSO buffer, a PIPES buffer,a PBS buffer, a MOPS buffer, an acetate buffer, a phosphate buffer, acacodylate buffer, a glycine buffer, a sulfate buffer, an imidazolebuffer, a guanidine hydrochloride buffer, a phosphate-citrate buffer, aborate buffer, a malonate buffer, a 3-picoline buffer, a 2-picolinebuffer, a 4-picoline buffer, a 3,5-lutidine buffer, a 3,4-lutidinebuffer, a 2,4-lutidine buffer, a Aces, a diethylmalonate buffer, aN-methylimidazole buffer, a 1,2-dimethylimidazole buffer, a TAPS buffer,a bis-Tris buffer, a L-arginine buffer, a lactate buffer, a glycolatebuffer.

In some embodiments, the PD-1 antibody and the second antibody areformulated in a buffer that is based on the buffer conditions of one ofthe two individual antibody formulations. In some embodiments, thebuffer conditions used are those of the anti-PD-1 antibody. In someembodiments, the anti-PD-1 antibody is nivolumab, and the two antibodiesare formulated in the citrate-based buffer system of nivolumab. In someembodiments, the buffer is a citrate buffer.

In some embodiments, the PD-1 antibody and the second antibody areformulated in buffer conditions that are different from the bufferconditions of either of the two antibodies on its own. In someembodiments, the buffer is a citrate-based buffer. In some embodiments,the concentration of citrate in the buffer is at least about 5 mM, about10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM,about 40 mM, or about 50 mM. In some embodiments, the concentration ofcitrate is between about 5 mM and about 50 mM, in some embodiments,between about 5 mM and about 40 mM, between about 5 mM and about 30 mM,between about 5 mM and about 20 mM, between about 5 mM and about 15 mM,between about 10 mM and about 30 mM, or between about 15 mM and about 25mM. In some embodiments, the concentration of citrate is about 10 mM. Insome embodiments, the concentration of citrate is about 20 mM.

In some embodiments, the buffer used is a Tris-based buffer. In someembodiments, the Tris buffer is a Tris-Cl buffer. In some embodiments,the concentration of Tris-Cl in the buffer is at least about 5 mM, about10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM,about 40 mM, or about 50 mM. In some embodiments, the concentration ofTris-Cl is between about 5 mM and about 50 mM, between about 10 mM andabout 50 mM, between about 10 mM and about 40 mM, between about 10 mMand about 30 mM or between about 15 mM and about 25 mM. In someembodiments, the concentration of Tris-Cl is about 20 mM.

In some embodiments, the buffer used is a histidine-based buffer. Insome embodiments, the concentration of histidine is at least about 5 mM,about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about35 mM, about 40 mM, or about 50 mM. In some embodiments, theconcentration of histidine is between about 5 mM and about 50 mM,between about 5 mM and about 40 mM, between about 5 mM and about 30 mM,between about 5 mM and about 25 mM or between about 10 mM and about 15mM. In some embodiments, the concentration of histidine is about 20 mM.

In some embodiments, the buffer used is a Tris-citrate buffer. In someembodiments, the concentration of Tris-Cl is at least about 5 mM, about10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM,about 40 mM, or about 50 mM and the concentration of citrate is at leastabout 2 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25mM, about 30 mM, about 35 mM, about 40 mM, or about 50 mM. In someembodiments, the concentration of Tris-Cl is between about 5 and about20 mM, between about 5 and about 15 mM, or between about 10 and about 15mM and the concentration of citrate is between about 1 mM and about 15mM, between about 1 mM and about 10 mM, or between about 5 mM and about10 mM. In some embodiments, the concentration of Tris-Cl is about 13.3mM and the concentration of citrate is about 6.7 mM.

In some embodiments, the pH of the composition is at least about 5,about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3,about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6,about 7.7, about 7.8, about 7.9, or about 8.0. In some embodiments, thepH of the composition is between about 5.0 and about 8.0, between about5.5 and about 6.5, between about 6.0 and about 7.0, or between about 6.5and about 7.5. In some embodiments, the pH is about 6.0, In otherembodiments the pH is about 7.0. In other embodiments, the pH is about6.2. In other embodiments, the pH is about 6.5. In other embodiments,the pH is about 6.6. In other embodiments, the pH is about 5.5.

In some embodiments, the composition of the invention further comprisesa bulking agent. A bulking agent can be selected from the groupconsisting of NaCl, mannitol, glycine, alanine, and any combinationthereof. In other embodiments, the composition of the inventioncomprises a stabilizing agent. The stabilizing agent can be selectedfrom the group consisting of sucrose, trehalose, raffinose, arginine; orany combination thereof. In other embodiments, the composition of theinvention comprises a surfactant. The surfactant can be selected fromthe group consisting of polysorbate 80 (PS80), polysorbate 20 (PS20),and any combination thereof. In certain embodiments, the compositionfurther comprises a chelating agent. The chelating agent can be selectedfrom the group consisting of diethylenetriaminepentaacetic acid (DTPA),ethylenediaminetetraacetic acid, nitrilotriacetic acid, and anycombination thereof.

In one embodiment, the composition comprises NaCl, mannitol, penteticacid (DTPA), sucrose, PS80, and any combination thereof. In anotherembodiment, the composition comprises NaCl at a concentration of atleast about 5 mM, at least about 10 mM, at least about 15 mM, at leastabout 20 mM, at least about 25 mM, at least about 30 mM, at least about35 mM, at least about 40 mM, at least about 45 mM, at least about 50 mM,at least about 60 mM, at least about 70 mM, at least about 75 mM, atleast about 80 mM, at least about 90 mM, at least about 100 mM, at leastabout 110 mM, at least about 120 mM, at least about 130 mM, at leastabout 140 mM, at least about 150 mM, at least about 175 mM, at leastabout 200 mM, at least about 225 mM, at least about 250 mM, at leastabout 275 mM, at least about 300 mM, at least about 350 mM, at leastabout 400 mM, at least about 450 mM or at least about 450 mM. In otherembodiments, the composition comprises between about 10 and about 200 mMNaCl, between about 25 and about 150 mM NaCl, between about 40 and about125 mM NaCl, between about 25 and about 75 mM NaCl, between about 50 andabout 100 mM NaCl or between about 75 and 125 mM NaCl. In someembodiments, the composition comprises about 100 mM NaCl. In certainembodiments, the composition comprises about 50 mM NaCl. In otherembodiments, the composition comprises about 83.3 mM NaCl. In yet otherembodiments, the composition comprises about 96.15 mM NaCl. In aparticular embodiment, the composition comprises about 78.57 mM NaCl.

In certain embodiments, the composition comprises mannitol (% w/v) USPat a concentration of at least about 0.25%, at least about 0.5%, atleast about 0.75%, at least about 1%, at least about 1.5%, at leastabout 2%, at least about 2.5%, at least about 3%, at least about 3.5%,at least about 4%, at least about 4.5%, at least about 5%, at leastabout 7.5% or at least about 10%. In other embodiments, the compositioncomprises between about 0.5% and about 5% mannitol, between about 0.5%and about 4% mannitol, between about 0.5% and about 1.5% mannitol,between about 1% and about 2% mannitol, or between about 2.5% and about3.5% mannitol. In yet other embodiments, the composition comprises about1% mannitol. In still other embodiments, the composition comprises about3.0% mannitol. In some embodiments, the composition comprises about1.67% mannitol. In certain embodiments, the composition comprises about1.15% mannitol. In a particular embodiment, the composition comprisesabout 1.86% mannitol.

In other embodiments, the composition comprises pentetic acid (DTPA),USP at a concentration of at least about 5 μM, at least about 10 μM, atleast about 15 μM, at least about 20 μM, at least about 25 μM, at leastabout 30 μM, at least about 40 μM, at least about 50 μM, at least about60 μM, at least about 70 μM, at least about 75 μM, at least about 80 μM,at least about 90 μM, at least about 100 μM, at least about 110 μM, atleast about 120 μM, at least about 130 μM, at least about 140 μM, atleast about 150 μM, at least about 175 μM, or at least about 200 μM. Insome embodiments, the composition comprises between about 10 μM andabout 200 μM DTPA, between about 10 μM and about 150 μM DTPA, betweenabout 10 μM and about 100 μM DTPA, between about 10 μM and about 30 μMDTPA, between about 50 μM and about 100 μM DTPA, or between about 75 μMand about 125 μM DTPA. In other embodiments, the composition comprisesDTPA at about 100 μM. In certain embodiments, the composition comprisesDTPA at about 20 μM. In yet other embodiments, the composition comprisesDTPA at about 73.3 μM. In a particular embodiment, the compositioncomprises DTPA at about 50 μM. In a specific embodiments, thecomposition comprises DTPA at about 93.85 μM. In certain embodiments,the composition comprises DTPA at about 65.71 μM.

In some embodiments, the composition comprises polysorbate 80, NF (PS80)(% w/v) at a concentration of at least about 0.005%, at least about0.01%, at least about 0.015%, at least about 0.02%, at least about0.03%, at least about 0.04%, at least about 0.05%, at least about 0.06%,at least about 0.07%, at least about 0.08%, at least about 0.09%, or atleast about 0.1%. In other embodiments, the composition comprisesbetween about 0.005% and about 0.1% PS80, between about 0.005% and about0.02% PS80, between about 0.005% and about 0.05% PS80, between about0.01% and about 0.02% PS80, between about 0.02% and about 0.1% PS80 orbetween about 0.01% and about 0.03% PS80. In still other embodiments,the composition comprises PS80 at a concentration of about 0.01%. In yetother embodiments, the composition comprises PS80 at a concentration ofabout 0.04%. In some embodiments, the composition comprises PS80 at aconcentration of about 0.013%. In a particular embodiment, thecomposition comprises PS80 at a concentration of about 0.05%. In someembodiments, the composition comprises PS80 at a concentration of about0.02%. In other embodiments, the composition comprises PS80 at aconcentration of about 0.012%. In a specific embodiment, the compositioncomprises PS80 at a concentration of about 0.23%.

In certain embodiments, the composition comprises sucrose (% w/v) at aconcentration of at least about 1%, at least about 2%, at least about3%, at least about 4%, at least about 4.5%, at least about 5%, at leastabout 5.5%, at least about 6%, at least about 6.5%, at least about 7%,at least about 7.5%, at least about 8%, at least about 8.5%, at leastabout 9%, at least about 9.5%, at least about 10%, at least about 12% orat least about 15% sucrose. In other embodiments, the compositioncomprises between about 1% and about 10%, between about 2% and about10%, between about 5% and about 10%, between about 5% and about 7%, orbetween about 7.5% and about 10% sucrose. In yet other embodiments, thecomposition comprises about 6% sucrose. In still other embodiments, thecomposition comprises about 8.5% sucrose. In other embodiments, thecomposition comprises about 8.0% sucrose.

In certain embodiments, the composition comprises nivolumab andipilimumab in a Tris-citrate buffer. In some embodiments the compositioncomprises a 1:1 ratio of nivolumab to ipilimumab in a buffer comprisingabout 13.3 mM Tris (or 13.3 mM Tris±10%, 20%, 30%, 40%, or 50%), about6.7 mM citrate, (or 6.7 mM citrate±10%, 20%, 30%, 40%, or 50%), about1.67% mannitol (1.67% mannitol±10%, 20%, 30%, 40%, or 50%), about 83.3mM NaCl (or 83.3 mM NaCl±10%, 20%, 30%, 40%, or 50%), about 73.3 μM DTPA(or 73.3 μM DTPA±10%, 20%, 30%, 40%, or 50%) and about 0.013% PS80 (or0.013% PS80±10%, 20%, 30%, 40%, or 50%) at a pH of about 6.2. In someembodiments the composition comprises a 3:1 ratio of nivolumab toipilimumab in a Tris-citrate buffer comprising about 1.15% mannitol (or1.15% mannitol±10%, 20%, 30%, 40%, or 50%), about 96.15 mM NaCl (or96.15 mM NaCl±10%, 20%, 30%, 40%, or 50%), about 93.85 DTPA (or 93.85 μMDTPA±10%, 20%, 30%, 40%, or 50%) and about 0.012% PS80 (or 0.012%PS80±10%, 20%, 30%, 40%, or 50%) at a pH of about 6.6. In someembodiments the composition comprises a 1:3 ratio of nivolumab toipilimumab in a Tris-citrate buffer comprising about 1.86% mannitol (or1.86% mannitol±10%, 20%, 30%, 40%, or 50%), about 78.57 mM NaCl (or78.57 mM NaCl±10%, 20%, 30%, 40%, or 50%), about 65.71 μM DTPA (or 65.71μM DTPA±10%, 20%, 30%, 40%, or 50%) and about 0.023% PS80 (or 0.023%PS80±10%, 20%, 30%, 40%, or 50%) at a pH of about 6.0.

In other embodiments, the composition comprises nivolumab and ipilimumabin a histidine buffer. In some embodiments the composition comprises a3:1 ratio of nivolumab to ipilimumab in a 20 mM histidine buffer (or 20mM histidine buffer±10%, 20%, 30%, 40%, or 50%) comprising about 50 mMNaCl (or 50 mM NaCl±10%, 20%, 30%, 40%, or 50%), about 50 μM DTPA (or 50μM DTPA±10%, 20%, 30%, 40%, or 50%), about 6% sucrose (or 6%sucrose±10%, 20%, 30%, 40%, or 50%), and about 0.05% PS80 (or 0.05%PS80±10%, 20%, 30%, 40%, or 50%) at about pH 6. In some embodiments thecomposition comprises a 3:1 ratio of nivolumab to ipilimumab in a about20 mM histidine buffer comprising about 50 mM NaCl (or 50 mM NaCl±10%,20%, 30%, 40%, or 50%), about 50 μM DTPA (or 50 μM DTPA±10%, 20%, 30%,40%, or 50%), about 6% sucrose (or 6% sucrose±10%, 20%, 30%, 40%, or50%), and about 0.05% PS80 (or 0.05% PS80±10%, 20%, 30%, 40%, or 50%) atabout pH 7. In some embodiments the composition comprises a 3:1 ratio ofnivolumab to ipilimumab in an about 20 mM histidine buffer (or 20 mMhistidine buffer±10%, 20%, 30%, 40%, or 50%) comprising about 50 μM DTPA(or 50 μM DTPA 10%, 20%, 30%, 40%, or 50%), about 8.5% sucrose (or 8.5%sucrose±10%, 20%, 30%, 40%, or 50%), and about 0.05% PS80 (or 0.05%PS80±10%, 20%, 30%, 40%, or 50%) at about pH 6. In some embodiments, thecomposition comprises a 1:1, 3:1, or 1:3 ratio of nivolumab toipilimumab in a histidine buffer (20 mM±10%, 20%, 30%, 40%, or 50%)comprising 5 μM DTPA (or 50 μM DTPA±10%, 20%, 30%, 40%, or 50%), 0.05%PS80 (or 0.05% PS80±10%, 20%, 30%, 40%, or 50%) and 8.0% sucrose (or8.0% sucrose±10%, 20%, 30%, 40%, or 50%) at pH 5.5, 6.0 or 6.5. In oneembodiment, the composition comprises nivolumab and ipilimumab in acitrate buffer. In another embodiment, the composition comprises a 3:1ratio of nivolumab to ipilimumab in an about 20 mM citrate buffer (or 20mM citrate buffer±10%, 20%, 30%, 40%, or 50%) comprising about 50 mMNaCl (or 50 mM NaCl±10%, 20%, 30%, 40%, or 50%), about 50 μM DTPA (or 50μM DTPA±10%, 20%, 30%, 40%, or 50%), about 6% sucrose (or 6%sucrose±10%, 20%, 30%, 40%, or 50%), and about 0.05% PS80 (or 0.05%PS80±10%, 20%, 30%, 40%, or 50%) at about pH 6. In other embodiments,the composition comprises a 3:1 ratio of nivolumab to ipilimumab in anabout 20 mM citrate buffer (or 20 mM citrate buffer±10%, 20%, 30%, 40%,or 50%) comprising about 50 mM NaCl (or 50 mM NaCl±10%, 20%, 30%, 40%,or 50%), about 20 μM DTPA (or 20 μM DTPA±10%, 20%, 30%, 40%, or 50%),about 3% mannitol (or 3% mannitol±10%, 20%, 30%, 40%, or 50%), and about0.04% PS80 (or 0.04% PS80±10%, 20%, 30%, 40%, or 50%) at about pH 6. Instill other embodiments, the composition comprises a 1:1 ratio ofnivolumab to ipilimumab in an about 20 mM citrate buffer (or 20 mMcitrate buffer±10%, 20%, 30%, 40%, or 50%) comprising about 50 mM NaCl(or 50 mM NaCl±10%, 20%, 30%, 40%, or 50%), about 100 μM DTPA (or 100 μMDTPA±10%, 20%, 30%, 40%, or 50%), about 3% mannitol (or 3% mannitol 10%,20%, 30%, 40%, or 50%), and about 0.02% PS80 (or 0.02% PS80±10%, 20%,30%, 40%, or 50%) at about pH 6. In certain embodiments, the compositioncomprises a 1:1 ratio of nivolumab to ipilimumab in an about 20 mMcitrate buffer (or 20 mM citrate buffer±10%, 20%, 30%, 40%, or 50%)comprising about 50 mM NaCl (or 50 mM NaCl±10%, 20%, 30%, 40%, or 50%),about 100 μM DTPA (or 100 μM DTPA±10%, 20%, 30%, 40%, or 50%), about 3%mannitol (or 3% mannitol±10%, 20%, 30%, 40%, or 50%), and about 0.02%PS80 (or 0.02% PS80±10%, 20%, 30%, 40%, or 50%) at about pH 6.5. In someembodiments, the composition comprises a 1:1 ratio of nivolumab toipilimumab in an about 20 mM citrate buffer (or 20 mM citratebuffer±10%, 20%, 30%, 40%, or 50%) comprising about 100 mM NaCl (or 100mM NaCl±10%, 20%, 30%, 40%, or 50%), about 100 μM DTPA (or 100 μMDTPA±10%, 20%, 30%, 40%, or 50%), about 1.0% mannitol (or 1.0%mannitol±10%, 20%, 30%, 40%, or 50%), and about 0.02% PS80 (or 0.02%PS80±10%, 20%, 30%, 40%, or 50%) at about pH 6.5. In yet otherembodiments, the composition comprises a 1:1 ratio of nivolumab toipilimumab in an about 20 mM citrate buffer (or 20 mM citratebuffer±10%, 20%, 30%, 40%, or 50%) comprising about 50 mM NaCl (or 50 mMNaCl±10%, 20%, 30%, 40%, or 50%), about 100 μM DTPA (or 100 μM DTPA±10%,20%, 30%, 40%, or 50%), about 6% sucrose (or 6% sucrose±10%, 20%, 30%,40%, or 50%), and about 0.02% PS80 (or 0.02% PS80±10%, 20%, 30%, 40%, or50%) at about pH 6.0.

In some embodiments, the composition comprises a 1:3 ratio of nivolumabto ipilimumab comprising about 4.62 mg/ml nivolumab, about 1.54 mg/mlipilimumab, about 18.5 mM Tris Hydrochloride, about 1.5 mM SodiumCitrate Dihydrate, about 96.2 mM NaCl, about 1.2% Mannitol, about 93.9μM Pentetic Acid, and about 0.012% PS80 at about pH 6.0.

In some embodiments, the composition comprises a 1:3 ratio of nivolumabto ipilimumab comprising about 4.61 mg/ml nivolumab, about 1.54 mg/mlipilimumab, about 18.46 mM Tris Hydrochloride, about 1.54 mM SodiumCitrate Dihydrate, about 96.15 mM NaCl, about 1.15% Mannitol, about93.85 μM Pentetic Acid, and about 0.012% PS80 at about pH 6.3.

In some embodiments, the pharmaceutical composition comprises 30 mg ofnivolumab and 90 mg of ipilimumab per vial. In other embodiments, thecomposition comprises 40 mg of nivolumab and 120 mg of ipilimumab pervial.

In other embodiments, the composition comprises a third antibody. Insome embodiments, the third antibody is any antibody disclosed herein.

Stability of the Compositions

In one embodiment, a composition disclosed herein is stable at about 0°C., about 5° C., about 10° C., about 15° C., about 20° C., about 25° C.,about 30° C., about 35° C., about 40° C., about 45° C., about 50° C., orabout 55° C. for at least about 1 week, at least about 2 weeks, at leastabout 1 month, at least about 2 months, at least about 3 months, atleast about 6 months, at least about 9 months, at least about 1 year, atleast about 2 years or at least about 5 years.

In another embodiment, composition exhibits a change of the acidic peak(e.g., deamidation) that is less than about 15%, about 14%, about 13%,about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%,about 5%, about 4%, about 3%, about 2% or about 1% after being storedfor about 1 month, about 2 months, about 3 months, about 4 months, about6 months, or about 1 year at about 5° C. In other embodiments,composition exhibits a change of the acidic peak that is less than about15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%,about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2% orabout 1% after being stored for about 1 month, about 2 months, about 3months, about 4 months, about 6 months, or about 1 year at about 25° C.In some embodiments, composition exhibits a change of the acidic peakthat is less than about 15%, about 14%, about 13%, about 12%, about 11%,about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%,about 3%, about 2% or about 1% after being stored for about 1 month,about 2 months, about 3 months, about 4 months, about 6 months, or about1 year at about 40° C. In some embodiments, the acidic peak is measuredusing an Imaged Capillary Isoelectric Focusing assay (cIEF).

In some embodiments, the deamidation of a composition of the presentinvention is comparable to the deamidation of a reference composition (acomposition comprising either the first antibody or the second antibody)if the composition exhibits a change of the acidic peak (e.g.,deamidation) that is less than about 15%, about 14%, about 13%, about12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about5%, about 4%, about 3%, about 2% or about 1% compared to the acidic peakof the reference composition.

In certain embodiments, the composition exhibits a change of the highmolecular weight (HMW) peak (e.g., aggregation) that is less than about15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%,about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2% orabout 1% after being stored for about 1 month, about 2 months, about 3months, about 4 months, about 6 months, or about 1 year at about 5° C.In some embodiments, the composition exhibits a change of the HMW peakthat is less than about 15%, about 14%, about 13%, about 12%, about 11%,about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%,about 3%, about 2% or about 1% after being stored for about 1 month,about 2 months, about 3 months, about 4 months, about 6 months, or about1 year at about 25° C. In some embodiments, the composition exhibits achange of the BMW peak that is less than about 15%, about 14%, about13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%,about 6%, about 5%, about 4%, about 3%, about 2% or about 1% after beingstored for about 1 month, about 2 months, about 3 months, about 4months, about 6 months, or about 1 year at about 40° C. In someembodiments, the composition exhibits a change of the HMW peak that isless than about 5%, about 4%, about 3%, about 2%, about 1.5%, about1.4%, about 1.3%, about 1.2%, about 1.1%, about 1%, about 0.9%, about0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about0.2%, about 0.1%, or less than about 0.1%. In certain embodiments, thecomposition exhibits a HMW peak that is about 15%, about 14%, about 13%,about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%,about 5%, about 4%, about 3%, about 2.5%, about 2%, about 1.5%, about1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about0.4%, about 0.3%, about 0.2% or about 0.1% after being stored for about1 month, about 2 months, about 3 months, about 4 months, about 6 months,or about 1 year at about 5° C., at about 25° C. or at about 40° C. Insome embodiments, the high molecular weight peak is measured usingchromatography. In some embodiments, the chromatography is sizeexclusion chromatography.

In some embodiments, the aggregation (e.g., a level of HMW species) of acomposition of the present invention is comparable to the aggregation ofa reference composition (a composition comprising either the firstantibody or the second antibody), if the composition exhibits a changeof the BMW species peak that is less than about 15%, about 14%, about13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%,about 6%, about 5%, about 4%, about 3%, about 2% or about 1% compared tothe HMW species peak of the reference composition.

In some embodiments, the composition exhibits a change of the main peakthat is less than about 15%, about 14%, about 13%, about 12%, about 11%,about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%,about 3%, about 2% or about 1% after being stored for about 1 month,about 2 months, about 3 months, about 4 months, about 6 months, or about1 year at about 5° C. In some embodiments, the composition exhibits achange of the main peak that is less than about 15%, about 14%, about13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%,about 6%, about 5%, about 4%, about 3%, about 2% or about 1% after beingstored for about 1 month, about 2 months, about 3 months, about 4months, about 6 months, or about 1 year at about 25° C. In someembodiments, the composition exhibits a change of the main peak that isless than about 15%, about 14%, about 13%, about 12%, about 11%, about10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about3%, about 2% or about 1% after being stored for about 1 month, about 2months, about 3 months, about 4 months, about 6 months, or about 1 yearat about 40° C. In some embodiments, the composition exhibits a changeof the main peak that is less than about 5%, about 4%, about 3%, about2%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about0.4%, about 0.3%, about 0.2%, or about 0.1%. In some embodiments, themain peak is measured using an Imaged Capillary Isoelectric Focusingassay (cIEF).

In some embodiments, the composition exhibits a change of the lowmolecular weight (LMW) peak (e.g., fragmentation) that is less thanabout 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%or about 1% after being stored for about 1 month, about 2 months, about3 months, about 4 months, about 6 months, or about 1 year at about 5° C.In some embodiments, the composition exhibits a change of the LMW peakthat is less than about 15%, about 14%, about 13%, about 12%, about 11%,about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%,about 3%, about 2% or about 1% after being stored for about 1 month,about 2 months, about 3 months, about 4 months, about 6 months, or about1 year at about 25° C. In some embodiments, the composition exhibits achange of the LMW peak that is less than about 15%, about 14%, about13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%,about 6%, about 5%, about 4%, about 3%, about 2% or about 1% after beingstored for about 1 month, about 2 months, about 3 months, about 4months, about 6 months, or about 1 year at about 40° C. In someembodiments, the composition exhibits a change of the LMW peak that isless than about 5%, about 4%, about 3%, about 2%, about 1.5%, about1.4%, about 1.3%, about 1.2%, about 1.1%, about 1%, about 0.9%, about0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about0.2%, or about 0.1%. In certain embodiments, the composition exhibits aLMW peak that is about 15%, about 14%, about 13%, about 12%, about 11%,about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%,about 3%, about 2.5%, about 2%, about 1.5%, about 1%, about 0.9%, about0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about0.2% or about 0.1% after being stored for about 1 month, about 2 months,about 3 months, about 4 months, about 6 months, or about 1 year at about5° C., at about 25° C. or at about 40° C. In some embodiments, the lowmolecular weight peak is measured using chromatography. In someembodiments, the chromatography is size exclusion chromatography.

In some embodiments, the fragmentation (e.g., a level of LMW species) ofa composition of the present invention is comparable to thefragmentation of a reference composition (a composition comprisingeither the first antibody or the second antibody), if the compositioncomprising the first and second antibodies exhibits a change of the LMWspecies peak that is less than about 15%, about 14%, about 13%, about12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about5%, about 4%, about 3%, about 2% or about 1% compared to the LMW speciespeak of the reference composition.

Method of Making the Compositions Disclosed Herein

In one embodiment, the invention is directed to a method of making anycomposition disclosed herein. In another embodiment, the formulationcomprising the anti-PD-1 antibody drug product is mixed with aformulation comprising the second antibody drug product to obtain thedesired ratio in a final drug product with no buffer changes. In otherembodiments, the final composition is in Tris-citrate buffer.

In some embodiments, the formulation comprising the anti-PD-1 antibodydrug substance and a formulation comprising the second antibody drugsubstance is subject to buffer exchanges and/or concentration beforebeing mixed to obtain the desired ratio in a final drug product.

In other embodiments, the composition is diluted prior to use. Incertain embodiments, the composition is diluted with 0.9% SodiumChloride Injection, USP or 5% Dextrose Injection, USP prior to use. Inother embodiments, the composition is diluted to obtain an infusion witha desired concentration of first and second antibody. In yet otherembodiments, the final concentration of first and second antibody isbetween about 1 mg/ml and about 500 mg/ml, about 1 mg/ml and about 450mg/ml, about 1 mg/ml and about 400 mg/ml, about 1 mg/ml and about 350mg/ml, about 1 mg/ml and about 300 mg/ml, about 1 mg/ml and about 250mg/ml, about 1 mg/ml and about 200 mg/ml, about 1 mg/ml and about 150mg/ml, about 1 mg/ml and about 100 mg/ml, about 1 mg/ml and about 90mg/ml, about 1 mg/ml and about 80 mg/ml, about 1 mg/ml and about 70mg/ml, about 1 mg/ml and about 60 mg/ml, about 1 mg/ml and about 50mg/ml, about 1 mg/ml and about 40 mg/ml, about 1 mg/ml and about 30mg/ml, about 1 mg/ml and about 20 mg/ml, about 1 mg/ml and about 15mg/ml, about 1 mg/ml and about 10 mg/ml, about 1 mg/ml and about 9mg/ml, about 1 mg/ml and about 8 mg/ml, about 1 mg/ml and about 7 mg/ml,about 1 mg/ml and about 6 mg/ml, about 1 mg/ml and about 5 mg/ml, about1 mg/ml and about 4 mg/ml, about 1 mg/ml and about 3 mg/ml, about 1mg/ml and about 2 mg/ml, about 0.5 mg/ml and about 3 mg/ml, about 50mg/ml and about 400 mg/ml, or about 100 mg/ml and about 300 mg/ml.

In certain embodiments, the diluted infusion is stored for no more thanabout 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3,about 2 or about 1 hours at room temperature after dilution. In someembodiments, the diluted infusion is store under refrigeration (about 2°C.-about 8° C.) for no more than about 1 week, about 6 days, about 5days, about 4 days, about 3 days, about 2 days, about 1 day or about 12hours after dilution.

Methods of the Invention

This disclosure provides a method of treating a subject afflicted with adisease or condition with any composition disclosed herein. In oneembodiment, the method is directed to administering a pharmaceuticalcomposition comprising an X amount of a first antibody, which is ananti-PD-1 antibody or an anti-PD-L1 antibody, and a Y amount of a secondantibody, wherein the ratio of the amount of the first antibody to theamount of the second antibody is present in a fixed dose ratio of about100:1 to about 1:100 in the composition.

In some embodiments, the disease or condition is an infectious disease.In other embodiments, the disease or condition is cancer. In still otherembodiments, the cancer is melanoma cancer, renal cancer, prostatecancer, breast cancer, colon cancer, lung cancer, bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous orintraocular malignant melanoma, uterine cancer, ovarian cancer, rectalcancer, cancer of the anal region, stomach cancer, testicular cancer,uterine cancer, carcinoma of the fallopian tubes, carcinoma of theendometrium, carcinoma of the cervix, carcinoma of the vagina, carcinomaof the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra, cancer of the penis, chronic or acute leukemias including acutemyeloid leukemia, chronic myeloid leukemia, acute lymphoblasticleukemia, chronic lymphocytic leukemia, solid tumors of childhood,lymphocytic lymphoma, cancer of the bladder, cancer of the kidney orureter, carcinoma of the renal pelvis, neoplasm of the central nervoussystem (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axistumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma,epidermoid cancer, squamous cell cancer, T-cell lymphoma,environmentally induced cancers including those induced by asbestos, orany combinations thereof. In yet other embodiments, the cancer is lungcancer, metastatic melanoma, glioblastoma, or renal cell carcinoma.

In certain embodiments, the cancer is squamous cell carcinoma,small-cell lung cancer, non-small cell lung cancer, squamous non-smallcell lung cancer (NSCLC), non-squamous NSCLC, glioma, gastrointestinalcancer, renal cancer (e.g. clear cell carcinoma), ovarian cancer, livercancer, colorectal cancer, endometrial cancer, kidney cancer (e.g.,renal cell carcinoma (RCC)), prostate cancer (e.g. hormone refractoryprostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreaticcancer, glioblastoma (glioblastoma multiforme), cervical cancer, stomachcancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, andhead and neck cancer (or carcinoma), gastric cancer, germ cell tumor,pediatric sarcoma, sinonasal natural killer, melanoma (e.g., metastaticmalignant melanoma, such as cutaneous or intraocular malignantmelanoma), bone cancer, skin cancer, uterine cancer, cancer of the analregion, testicular cancer, carcinoma of the fallopian tubes, carcinomaof the endometrium, carcinoma of the cervix, carcinoma of the vagina,carcinoma of the vulva, cancer of the esophagus, cancer of the smallintestine, cancer of the endocrine system, cancer of the parathyroidgland, cancer of the adrenal gland, sarcoma of soft tissue, cancer ofthe urethra, cancer of the penis, solid tumors of childhood, cancer ofthe ureter, carcinoma of the renal pelvis, neoplasm of the centralnervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinalaxis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma,epidermoid cancer, squamous cell cancer, T-cell lymphoma,environmentally-induced cancers including those induced by asbestos,virus-related cancers (e.g., human papilloma virus (HPV)-related tumor),and hematologic malignancies derived from either of the two major bloodcell lineages, i.e., the myeloid cell line (which produces granulocytes,erythrocytes, thrombocytes, macrophages and mast cells) or lymphoid cellline (which produces B, T, NK and plasma cells), such as all types ofleukemias, lymphomas, and myelomas, e.g., acute, chronic, lymphocyticand/or myelogenous leukemias, such as acute leukemia (ALL), acutemyelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), andchronic myelogenous leukemia (CML), undifferentiated AML (M0),myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cellmaturation), promyelocytic leukemia (M3 or M3 variant [M3V]),myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]),monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia(M7), isolated granulocytic sarcoma, and chloroma; lymphomas, such asHodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B cellhematologic malignancy, e.g., B-cell lymphomas, T-cell lymphomas,lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma,mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki1+) large-cell lymphoma, adult T-cell lymphoma/leukemia, mantle celllymphoma, angio immunoblastic T-cell lymphoma, angiocentric lymphoma,intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma,precursor T-lymphoblastic lymphoma, T-lymphoblastic; andlymphoma/leukaemia (T-Lbly/T-ALL), peripheral T-cell lymphoma,lymphoblastic lymphoma, post-transplantation lymphoproliferativedisorder, true histiocytic lymphoma, primary central nervous systemlymphoma, primary effusion lymphoma, lymphoblastic lymphoma (LBL),hematopoietic tumors of lymphoid lineage, acute lymphoblastic leukemia,diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma,diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma,precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC)(also called mycosis fungoides or Sezary syndrome), andlymphoplasmacytoid lymphoma (LPL) with Waldenstrom's macroglobulinemia;myelomas, such as IgG myeloma, light chain myeloma, nonsecretorymyeloma, smoldering myeloma (also called indolent myeloma), solitaryplasmocytoma, and multiple myelomas, chronic lymphocytic leukemia (CLL),hairy cell lymphoma; hematopoietic tumors of myeloid lineage, tumors ofmesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma;seminoma, teratocarcinoma, tumors of the central and peripheral nervous,including astrocytoma, schwannomas; tumors of mesenchymal origin,including fibrosarcoma, rhabdomyoscaroma, and osteosarcoma; and othertumors, including melanoma, xeroderma pigmentosum, keratoacanthoma,seminoma, thyroid follicular cancer and teratocarcinoma, hematopoietictumors of lymphoid lineage, for example T-cell and B-cell tumors,including but not limited to T-cell disorders such as T-prolymphocyticleukemia (T-PLL), including of the small cell and cerebriform cell type;large granular lymphocyte leukemia (LGL) preferably of the T-cell type;a/d T-NHL hepatosplenic lymphoma; peripheral/post-thymic T cell lymphoma(pleomorphic and immunoblastic subtypes); angiocentric (nasal) T-celllymphoma; cancer of the head or neck, renal cancer, rectal cancer,cancer of the thyroid gland; acute myeloid lymphoma, as well as anycombinations of said cancers. The methods described herein can also beused for treatment of metastatic cancers.

In certain embodiments, the composition is administered with anyadditional anti-cancer agent. In other embodiments, the anti-canceragent is any anti-cancer agent that is known in the art. In yet otherembodiments, the anti-cancer agent is a third antibody. In someembodiments, the third antibody is any antibody disclosed herein.

In other embodiments, the composition is administered intravenously. Insome embodiments, the composition is reconstituted prior toadministration. In yet other embodiments, the composition is dilutedprior to administration. In a particular embodiment, the composition isadministered at a flat dose. In other embodiments, the composition isadministered at a weight-based dose.

In some embodiments, the composition is administered at least aboutweekly, at least about twice weekly, at least about every two weeks, atleast about every three weeks, or at least about monthly. In someembodiments, the treatment lasts for at least about 4 weeks, at leastabout 8 weeks, at least about 12 weeks, at least about 3 months, atleast about 6 months, at least about 9 months, at least about 1 year, atleast about 18 months, at least about 2 years or greater than 2 years.

In some embodiments, the invention is directed to a method of modulatingthe immune response comprising administering any composition disclosedherein.

In certain embodiments, the composition of the present invention (e.g.,administration of an anti-PD-1 antibody or the administration of ananti-PD-1 antibody and another anti-cancer therapy) effectivelyincreases the duration of survival of the subject. For example, theduration of survival of the subject is increased by at least about 1month, at least about 2 months, at least about 3 months, at least about4 months, at least about 5 months, at least about 6 months, at leastabout 7 months, at least about 8 months, at least about 9 months, atleast about 10 months, at least about 11 months or at least about 1 yearor more when compared to another subject treated with only eitheranother therapy (e.g., the standard of care) or only one of the twomembers of the composition alone (e.g., an anti-PD-1 antibody alone). Insome embodiments, the duration of survival is increased by at leastabout 2 months. In certain embodiments, the therapy of the presentinvention effectively increases the duration of progression-freesurvival of the subject. For example, the progression free survival ofthe subject is increased by at least about 1 month, at least about 2months, at least about 3 months, at least about 4 months, at least about5 months, at least about 6 months, at least about 7 months, at leastabout 8 months, at least about 9 months, at least about 10 months, atleast about 11 months or at least about 1 year when compared to anuntreated subject or a subject treated only with another therapy (e.g.,standard of care treatment) or only one of the two members of thecomposition alone (e.g., an anti-PD-1 or PD-L1 antibody alone). In someembodiments, the progression-free survival is increased by at leastabout 2 months. In certain embodiments, the therapy of the presentinvention effectively increases the response rate in a group ofsubjects. For example, the response rate in a group of subjects isincreased by at least about 2%, at least about 3%, at least about 4%, atleast about 5%, at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at last about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 55%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 99% or at least about 100% when compared to another groupof subjects treated with only either another therapy (e.g., the standardof care) or only one of the two members of the composition alone (e.g.,an anti-PD-1 antibody alone), i.e., monotherapy.

Dosages of Compositions Disclosed Herein

In some embodiments, the composition is administered at a flat doseregardless of the weight of the patient. For example, the anti-PD-1antibody with the second antibody may be administered at a flat dose of0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 50, 75, 80, 200, 240, 300, 360,400, 480, 500, 750 or 1500 mg or any other dose disclosed herein,without regard to the patient's weight. In some embodiments thecomposition is administered at a weight-based dose at any dose disclosedherein. In some embodiments, the amount of the first antibody and theamount of the second antibody administered to the patient at a singledose are identical the X amount and the Y amount, respectively.

In certain embodiments of the present combination therapy methods, thetherapeutically effective dosage of the anti-PD-1 antibody orantigen-binding portion thereof comprises 60 mg, about 80 mg, about 100mg, about 120 mg, about 140, about 160 mg, about 180 mg, about 200 mg,about 220 mg, about 240 mg, about 260 mg, about 280 mg, or about 300 mg.In some embodiments, the therapeutically effective dosage of theanti-PD-1 antibody or antigen-binding portion thereof comprises about310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about460 mg, about 470 mg, about 480 mg, about 490 mg, or about 500 mg. Insome embodiments, the dose of the anti-PD-1 antibody in the compositionis between about 60 mg and about 300 mg, between about 60 mg and about100 mg, between about 100 mg and about 200 mg, or between about 200 mgand about 300 mg. In some embodiments, the dose of the anti-PD-1antibody in the composition is between about 300 mg and about 500 mg,between about 300 mg and about 450 mg, between about 300 mg and about400 mg, between about 300 mg and about 350 mg, between about 350 mg andabout 500 mg, between about 400 mg and about 500 mg, or between about450 mg and about 500 mg. In some embodiments, the amount of theanti-PD-1 antibody in the composition is at least about 80 mg, about 160mg, or about 240 mg. In certain embodiments, the amount of the anti-PD-1antibody in the composition is at least about 360 mg or 480 mg. In someembodiments, the dose of the anti-PD-1 antibody in the composition is atleast about 240 mg or at least about 80 mg. In one embodiment, theamount of the anti-PD-1 antibody in the composition is about 360 mg. Inanother embodiment, the amount of the anti-PD-1 antibody in thecomposition is about 480 mg. In some embodiments, the dose of theanti-PD-1 antibody in the composition is a least about 0.5 mg/kg, atleast about 1 mg/kg, at least about 2 mg/kg, at least about 3 mg/kg orat least about 5 mg/kg. In some embodiments, the dose of anti-PD-1antibody in the composition is between about 0.5 mg/kg and about 5mg/kg, between about 0.5 mg/kg and about 5 mg/kg, between about 0.5mg/kg and about 3 mg/kg or between about 0.5 mg/kg and about 2 mg/kg. Insome embodiments, the dose of the anti-PD-1 antibody in the compositionis a least about 1 mg/kg. The corresponding dose of the second antibodyis calculated using the desired ratio.

In some embodiments, the anti-PD-1 antibody is administered at asubtherapeutic dose, i.e., a dose of the therapeutic agent that issignificantly lower than the usual or FDA-approved dose whenadministered as monotherapy for the treatment of the cancer. Thequantity of the second antibody in the composition is calculated basedon the desired ratio. Dosages of nivolumab that are lower than thetypical 3 mg/kg, but not less than 0.001 mg/kg, are subtherapeuticdosages. The subtherapeutic doses of an anti-PD-1 antibody used in themethods herein are higher than 0.001 mg/kg and lower than 3 mg/kg. Insome embodiments, a subtherapeutic dose is about 0.001 mg/kg-about 1mg/kg, about 0.01 mg/kg-about 1 mg/kg, about 0.1 mg/kg-about 1 mg/kg, orabout 0.001 mg/kg-about 0.1 mg/kg body weight. In some embodiments, thesubtherapeutic dose is at least about 0.001 mg/kg, at least about 0.005mg/kg, at least about 0.01 mg/kg, at least about 0.05 mg/kg, at leastabout 0.1 mg/kg, at least about 0.5 mg/kg, or at least about 1.0 mg/kgbody weight. Receptor-occupancy data from 15 subjects who received 0.3mg/kg to 10 mg/kg dosing with nivolumab indicate that PD-1 occupancyappears to be dose-independent in this dose range. Across all doses, themean occupancy rate was 85% (range, 70% to 97%), with a mean plateauoccupancy of 72% (range, 59% to 81%). In some embodiments, 0.3 mg/kgdosing can allow for sufficient exposure to lead to maximal biologicactivity.

In some embodiments, the composition is administered by intravenousinfusion once about per week, once about every 2 weeks, once about every3 weeks, or once about a month. In certain embodiments, the compositionis administered once about every 3 weeks. In one embodiment, 360 mg ofthe anti-PD-1 antibody or antigen binding fragment is administered onceevery 3 weeks. In another embodiment, 480 mg of the anti-PD-1 antibodyor antigen binding fragment is administered once about once every 4weeks. In some embodiments, the infusion occurs over at least about 10minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 60minutes, about 90 minutes, about 2 hours, about 3 hours, about 4 hoursor about 5 hours.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention can be flat or varied so as toobtain an amount of the active ingredient which is effective to achievethe desired therapeutic response for a particular patient, composition,and mode of administration, without being unduly toxic to the patient.The selected dosage level will depend upon a variety of pharmacokineticfactors including the activity of the particular compositions of thepresent invention employed, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular compositions employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors well known in themedical arts. A composition of the present invention can be administeredvia one or more routes of administration using one or more of a varietyof methods well known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results.

Kits

Also within the scope of the present invention are kits comprising ananti-PD-1 antibody/second antibody compositions and instructions fortherapeutic uses. Kits typically include a label indicating the intendeduse of the contents of the kit and instructions for use. The term labelincludes any writing, or recorded material supplied on or with the kit,or which otherwise accompanies the kit. Accordingly, this disclosureprovides a kit comprising: (a) an appropriate dosage of the compositiondisclosed herein and (b) instructions for using the composition in anyof the methods disclosed herein.

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting. The contents of allreferences cited throughout this application are expressly incorporatedherein by reference.

EXAMPLES

Several feasibility studies were performed to evaluate the stability ofipilimumab and nivolumab in a single fixed dose ratio combination (FDRC)formulation. FIG. 1 shows the formulations of ipilimumab and nivolumabin their drug substance (DS) or drug product (DP) formulations, whichwere used as controls where indicated in the following examples.

Example 1

A feasibility study was performed to evaluate the stability ofipilimumab and nivolumab in a single fixed dose ratio combination (FDRC)formulation created by mixing the individual formulations of ipilimumaband nivolumab (FIG. 1) to a final ratio of ipilimumab to nivolumab of1:1.

Ipilimumab (BMS-734016) DP contains 5 mg/mL ipilimumab in 20 mMTris-HCl, 100 mM NaCl, 1.0% (w/v) Mannitol, 100 μM pentetic acid (DTPA),and 0.01% polysorbate 80 (PS80), at pH 7.0, and it is available as 40 mLin a 50 mL bottle and 10 ml in a 10 ml vial (FIG. 1). Nivolumab(BMS-936558) DP contains 10 mg/mL nivolumab in 20 mM citrate buffer(sodium citrate dihydrate), 50 mM NaCl, 3.0% (w/v) Mannitol, 20 μM DTPA,and 0.02% PS80, at pH 6.0, and it is available as 10 mL in a 10 ml vial(FIG. 1).

To achieve a 1:1 ratio of ipilimumab to nivolumab, 80 mL of ipilimumabDP (2 bottles) was mixed with 40 mL of nivolumab DP (4 vials), yieldinga combined product having 3.3 mg/mL ipilimumab and 3.3 mg/mL nivolumab.The resulting FDRC formulation contained 13.3 mM Tris-HCl, 6.7 mMcitrate, 83.3 mM NaCl, 1.67% (w/v) mannitol, 73.3 μM DTPA, and 0.013%w/v PS80, at pH 6.2 as shown in Table 1.

TABLE 1 Combined EC FDRC (1:1) Formulation Final Conc'n in Vial: (mg/mL)Tris Citrate Mannitol NaCl DTPA PS 80 Prototype Ratio Ipi (mg/mL) Nivo(mg/mL) mM mM pH % w/v mM μM % w/v 1 1:1 3.3 3.3 13.3 6.7 6.2 1.67 83.373.3 0.013

The FDRC (1:1) formulation was filtered and aliquoted into 10 cc glassvials (5 mL per vial), stoppered, and sealed. Vials were then stored ateither 5° C. or 40° C. Samples were analyzed at 0 days, 1 week, 2 weeks,1 month, 2 months, 3 months, and 6 months. Day-0 samples were used acontrols.

Sample Analysis—Methods

At each time point, sample vials were analyzed by visual appearance, pHat room temperature, HIAC, size exclusion chromatography, and imagedcapillary isoelectric focusing (cIEF). HIAC (Royco) is a lightobscuration based particle counting technique instrument.

Size exclusion chromatography (SEC) was performed by analytical sizeexclusion HPLC (SE-HPLC) using a TSKGEL® G3000SW_(XL) with a TSKGEL®Guard SW_(XL) guard column on a WATERS® 2695 ALLIANCE® HPLC with a 2497dual wavelength UV detector using EMPOWER™ 2 Software. The system wasequilibrated with 0.1 M NaH₂PO₄, 0.1 M Na₂SO₄, and 15% acetonitrile(ACN), at pH 6.8 (mobile phase). Samples were analyzed neat unless theconcentration was greater than 125 mg/mL. If the sample concentrationwas greater than 125 mg/mL, the sample was diluted to 50 mg/mL with thecorresponding buffer. Samples were transferred to an HPLC vial prior toanalysis and stored in the analytical HPLC system at a temperature of 5°C.±3° C. A total of 100 μg of sample were injected for analysis and wererun isocratically with a column temperature of 22° C. using the mobilephase. The flow rate was 1.0 mL/min with a run time per sample of 20 minand a detection wavelength of 280 nm.

Imaged capillary isoelectric focusing (cIEF) was performed using aProtein SIMPLE™ iCE3 instrument with an Alcott sampler. Samples wereanalyzed at a concentration of 25 mg/mL with 2 M urea and 0.35% methylcellulose (MC). A 50 mm capillary with a 100 μm internal diameter wasused to perform separation. The electrolyte solution was 80 mM H₃PO₄ in0.1% MC, and the catholyte solution was 100 mM NaOH in 0.1% MC. Thecarrier ampholytes were 1% PHARMALYTE® 5-8 and 3% PHARMALYTE® 8-10.5.Focusing time was 13 minutes with focusing voltage starting at 1.5 kV(300 V/cm) for the first minute followed by 3 kV (600V/cm) for theremaining 12 minutes. Detection was performed at 280 nm.

Sample Analysis—Results

SEC was performed on the nivolumab DP and ipilimumab DP controls and the1:1 ratio fixed dosing combination (EC FDRC (1:1)) formulation followingstorage for 3 months at 40° C. (FIG. 2A). The nivolumab DP control BMWpeak size increased by about 1.6%, and the ipilimumab DP control HMWpeak size increased by about 0.25% following storage for 3 months at 40°C. (FIG. 2A). The EC FDRC (1:1) formulation HMW peak size increased byabout 0.7% following storage for 3 months at 40° C. (FIG. 2A). Followingstorage for 6 months at 40° C., the EC FDRC (1:1) formulation HMW peaksize increased from 0.555% to a final HMW peak size of 2.82%, anincrease of about 2.265% (Table 2). Following storage for 6 months at 5°C., the EC FDRC (1:1) formulation HMW peak size decreased from 0.555% toa final HMW peak size of 0.525% (Table 2).

TABLE 2 Combined EC FDRC (1:1) Capillary Isoelectric Focusing and SizeExclusion Chromatography results. cIEF Acidic cIEF Acidic cIEF AcidicSEC HMW SEC HMW Peak Peak Peak SEC HMW SEC HMW SEC HMW Change at Changeat Drug (Initial) (6 M/5 C.) (Change at 5 C.) Initial 6 M/40 C. 6 M/5 C.40 C. 5 C. Ipilimumab 34 38.16 4.16 0.555 2.82 0.525 2.265 −0.03Nivolumab 35.09 38.65 3.56

cIEF was performed on the nivolumab DP and ipilimumab DP controls andthe EC FDRC (1:1) formulation following storage for 6 months at 5° C.(FIG. 2B). The nivolumab DP control acidic peak size increased by about13%, and the ipilimumab DP acidic peak size increased by about 3%following storage for 6 months at 5° C. (FIG. 2B). For the EC FDRC (1:1)formulation, the nivolumab acidic peak size increased by about 3.56%,from 35.09% at day-0 (Initial) to 38.65% at 6 months, while theipilimumab acidic peak size increased by about 4.16%, from 34% at day-0(Initial) to 38.16% at 6 months (Table 2 and FIG. 2B).

This study can be used to leverage a broad concentration range of amixed buffer system, i.e., a Tris-Citrate buffer composition.

Example 2

A feasibility study was performed to evaluate the stability of anipilimumab/nivolumab FDRC created by mixing the individual formulationsof ipilimumab and nivolumab to final ratios of 3:1, 1:1, and 1:3 (Table3). The FDRC formulations were generated by mixing the ipilimumab DS at5 mg/mL and nivolumab DS at 20 mg/mL to achieve 3:1, 1:1, and 1:3protein ratios (Table 3). Each combined solution was further mixed witha stir bar at room temperature for 30 min, transferred to vials, andstored for stability over time. The vials were stored at 5° C., 25° C.,and 40° C. for up to 12 months.

TABLE 3 EC FDRC (3:1; 1:1; 1:3) - Combinations of Formulations ofIpilimumab DP and Nivolumab DP Final Conc'n in Vial: (mg/mL) MannitolNaCl DTPA PS 80 Prototype Ratio Ipi Nivo pH % w/v mM μM % w/v EC: pH 6.63:1 4.62 1.54 6.6 1.15 96.15 93.85 0.012 EC: pH 6.0 1:3 2.86 8.57 6.01.86 78.57 65.71 0.023 EC: pH 6.2 1:1 4.00 4.00 6.2 1.67 83.33 73.330.013

Prototype EC: pH 6.6, having a 3:1 ratio of ipilimumab to nivolumab,contained 4.62 mg/mL ipilimumab, 1.54 mg/mL nivolumab, 1.15% w/vmannitol, 96.15 mM NaCl, 93.85 μM DTPA, and 0.012% w/v PS80, at pH 6.6.Prototype EC: pH 6.0, having a 1:3 ratio of ipilimumab to nivolumab,contained 2.86 mg/mL ipilimumab, 8.57 mg/mL nivolumab, 1.86% w/vmannitol, 78.57 mM NaCl, 65.71 μM DTPA, and 0.023% w/v PS80, at pH 6.0.Prototype EC: pH 6.2, having a 1:1 ratio of ipilimumab to nivolumab,contained 4.00 mg/mL ipilimumab, 4.00 mg/mL nivolumab, 1.67% w/vmannitol, 83.33 mM NaCl, 73.33 μM DTPA, and 0.013% w/v PS80, at pH 6.2.

SEC Analysis

In general, small increases in HMW and LMW were observed in all 3prototypes (FIGS. 3A-B). SEC was performed on the nivolumab DP control,the ipilimumab DP control, and the EC FDRC formulations EC: pH 6.0(1:3), EC: pH 6.2 (1:1), and EC: pH 6.6 (3:1) following storage for 2months at 40° C. (FIGS. 3A and 3B). The ipilimumab control formulationhad an initial HMW peak size of about 0.4% at day 0, which increased byabout 0.1% to a final HMW peak size of just over 0.5% after 2 months at40° C. (FIG. 3A). The nivolumab control formulation had an initial BMWpeak size of about 0.8% at day 0, which increased by about 0.7% to afinal HMW peak size of over 1.5% after 2 months at 40° C. (FIG. 3A). TheEC: pH 6.0 FDRC formulation (1:3) had an initial BMW peak size of about0.6% at day 0, which increased by about 0.7% to a final BMW peak size ofabout 1.3% after 2 months at 40° C. (FIG. 3A). The EC: pH 6.2 FDRCformulation (1:1) had an initial HMW peak size of about 0.5% at day 0,which increased by about 0.5% to a final HMW peak size of about 1.0%after 2 months at 40° C. (FIG. 3A). The EC: pH 6.6 FDRC formulation(3:1) had an initial BMW peak size of about 0.5% at day 0, whichincreased by about 0.3% to a final HMW peak size of about 0.8% after 2months at 40° C. (FIG. 3A).

The low molecular weight (LMW) peak sizes for the various formulationswere also measured at day 0, after 2 months at 40° C., and after 3months at 25° C. (FIG. 3B). The ipilimumab control formulation had aninitial LMW peak size of about 0.2% at day 0, which increased by about0.65% to a final LMW peak size of about 0.85% after 2 months at 40° C.(FIG. 3B). Following storage at 25° C. for 3 months, the LMW peak sizeof the ipilimumab control formulation increased by about 0.1% (FIG. 3B).The nivolumab control formulation had an initial LMW peak size of about0.2% at day 0, which increased by about 0.6% to a final LMW peak size ofabout 0.8% after 2 months at 40° C. (FIG. 3B). Following storage at 25°C. for 3 months, the LMW peak size of the nivolumab control formulationincreased by less than 0.1% (FIG. 3B). The EC: pH 6.0 FDRC formulation(1:3) had an initial LMW peak size of about 0.15% at day 0, whichincreased by about 0.8% to a final LMW peak size of about 0.95% after 2months at 40° C. (FIG. 3B). Following storage at 25° C. for 3 months,the LMW peak size of the EC: pH 6.0 (1:3) FDRC formulation increased byabout 0.2% (FIG. 3B). The EC: pH 6.2 FDRC formulation (1:1) had aninitial LMW peak size of about 0.15% at day 0, which increased by about1.2% to a final LMW peak size of about 1.35% after 2 months at 40° C.(FIG. 3B). Following storage at 25° C. for 3 months, the LMW peak sizeof the EC: pH 6.2 (1:1) FDRC formulation increased by about 0.3% (FIG.3B). The EC: pH 6.6 FDRC formulation (3:1) had an initial LMW peak sizeof about 0.15% at day 0, which increased by about 1.5% to a final LMWpeak size of about 1.65% after 2 months at 40° C. (FIG. 3B). Followingstorage at 25° C. for 3 months, the LMW peak size of the EC: pH 6.6(3:1) FDRC formulation increased by about 0.1%.

cIEF Analysis

cIEF was performed on the nivolumab DP control, the ipilimumab DPcontrol, and the EC FDRC formulations EC: pH 6.0 (1:3), EC: pH 6.2(1:1), and EC: pH 6.6 (3:1) following storage for 3 months at 25° C.(FIG. 4A), 3 months at 5° C. (FIG. 4B), and 1 month at 25° C. (FIG. 4C).The nivolumab DP control acidic peak size decreased by about 0.05% andthe ipilimumab DP control acidic peak size increased by about 5.59%following storage for 3 months at 25° C. (FIG. 4A). The FDRC formulationEC: pH 6.0 (1:3) nivolumab and ipilimumab acidic peak sizes increased byabout 5% and about 5.7%, respectively, following storage for 3 months at25° C. (FIG. 4A). The FDRC formulation EC: pH 6.2 (1:1) nivolumab andipilimumab acidic peak sizes increased by about 6.8% and about 6.3%,respectively, following storage for 3 months at 25° C. (FIG. 4A). TheFDRC formulation EC: pH 6.6 (3:1) nivolumab and ipilimumab acidic peaksizes increased by about 4% and about 7.8%, respectively, followingstorage for 3 months at 25° C. (FIG. 4A). Across the three FDRCformulations, the ipilimumab acidic peak size increased by about5.7%-7.8%, or by an average of about 2.2% per month; and the nivolumabacidic peak size increased by about 4%-6.8%, or by an average of lessthan 2% (about 1.76%) per month (FIG. 4A).

FIG. 4B shows the actual change in acidic peak size relative to theinitial (Day 0) controls for samples stored for 3 months at 5° C. usingcIEF analysis. The nivolumab DP control acidic peak size decreased byabout 5.1%, and the ipilimumab DP control acidic peak size decreased byabout 1% following storage for 3 months at 5° C. (FIG. 4B). The FDRCformulation EC: pH 6.0 (1:3) nivolumab and ipilimumab acidic peak sizesincreased by about 0.1% and decreased by about 1.5%, respectively,following storage for 3 months at 5° C. (FIG. 4B). The FDRC formulationEC: pH 6.2 (1:1) nivolumab and ipilimumab acidic peak sizes increased byabout 2.1% and about 0.5%, respectively, following storage for 3 monthsat 5° C. (FIG. 4B). The FDRC formulation EC: pH 6.6 (3:1) showed nochange in the ipilimumab acidic peak size and a decrease of less than0.1% in the nivolumab acidic peak size following storage for 3 months at5° C. (FIG. 4B).

FIG. 4C shows the actual change in acidic peak size relative to theinitial (Day 0) controls for samples stored for 1 month at 25° C. Thenivolumab DP control acidic peak size increased by about 1.05%, and theipilimumab DP control acidic peak size increased by about 1.16%following storage for 1 month at 25° C. (FIG. 4C). The FDRC formulationEC: pH 6.0 (1:3) nivolumab and ipilimumab acidic peak sizes increased byabout 2.8% and about 1%, respectively, following storage for 1 month at25° C. (FIG. 4C). The FDRC formulation EC: pH 6.2 (1:1) nivolumab andipilimumab acidic peak sizes increased by about 3.1% and about 1.6%,respectively, following storage for 1 month at 25° C. (FIG. 4C). TheFDRC formulation EC: pH 6.6 (3:1) nivolumab acidic peak size did notchange, and the ipilimumab acidic acid peak size increased by about 2.8%following storage for 1 month at 25° C. (FIG. 4C).

Example 3

A design of experiments (DoE) study was performed to identify newcandidate ipilimumab/nivolumab formulations. Prototypeipilimumab/nivolumab FDRC (3:1) formulations were made in selectedhistidine or citrate formulations, as shown in Table 4. All DoE FDRCprototypes were prepared to a final concentration ofipilimumab/nivolumab of 10 mg/mL at a ratio of 3:1 (Table 4). FDRCprototype “Combo 4” contained 20 mM citrate, 50 mM NaCl, 50 μM DTPA, 6%w/v sucrose, and 0.05% w/v PS80, at a theoretical pH of 6. FDRCprototype “Combo 5” contained 20 mM histidine, 50 mM NaCl, 50 μM DTPA,6% w/v sucrose, and 0.05% w/v PS80, at a theoretical pH of 6.0. FDRCprototype “Combo 6” contained 20 mM histidine, 50 mM NaCl, 50 μM DTPA,6% w/v sucrose, and 0.05% w/v PS80, at a theoretical pH of 7. FDRCprototype “Combo New” contained 20 mM histidine, 50 μM DTPA, 8.5% w/vsucrose, and 0.05% w/v PS80, at a theoretical pH of 6. FDRC prototype“Combo 8,” which was similar to the current nivolumab DP formulation,contained 20 mM citrate, 50 mM NaCl, 20 μM DTPA, 3% w/v mannitol, and0.04% w/v PS80, at a theoretical pH of 6.

TABLE 4 DoE FDRC (3:1) - Novel Formulations Prototype 20 mMConcentration NaCl DTPA Sucrose Mannitol PS80 (3:1) pH Buffer mg/mL mMμM % w/v % w/v % w/v Combo 4 6 Citrate 10 50 50 6 — 0.05 Combo 5 6Histidine 10 50 50 6 — 0.05 Combo 6 7 Histidine 10 50 50 6 — 0.05 Combo6 Histidine 10 — 50 8.5 — 0.05 new Combo 8 6 Citrate 10 50 20 — 3 0.04

The DoE FDRC formulations were created according to the followingexample preparation of Combo New. Combo New was prepared by firstsubjecting ipilimumab DS and nivolumab DS (ELN 96488-024 and -025) toultrafiltration/diafiltration. In particular, a disposable UFDF cassettewas used for nivolumab DS and ipilimumab DS. About 250 mL of theunformulated DS of nivolumab (˜21 mg/mL) was used for UF/DF, using thediafiltration/concentration mode. The transmembrane pressure (TMP) wasset at 15 psi, while a 0.3 liter per min flow speed was set for feedpump. The diafiltration was completed after 3 liter of the buffer wasused. The sample in the vessel was further concentrated based on a scaleweight reduction, and collected in 250 PETG bottle. The concentration ofnivolumab post UFDF was 30.6 mg/mL. About 500 mL of unformulated DS ofipilimumab (˜5.2 mg/mL) was used for UF/DF, using thediafiltration/concentration mode. The ipilimumab concentration in thefinal product was 16.2 mg/mL by A280.

Next, 20 mL of ipilimumab DS in a histidine-sucrose based buffer and 7.5mL of nivolumab DS in a histidine-sucrose based buffer were added intoD-Tube Dialyzer units and dialyzed against Combo New buffer, as shown inTable 4, for 24 hours in a cold room with sufficient volume (3× changeof buffer). The protein concentration of the ipilimumab and nivolumabwas then measured by HIAC. Additional ipilimumab DS and/or nivolumab DSand the appropriate buffers were then added to bring the finalconcentration of ipilimumab to 7.5 mg/mL and nivolumab to 2.5 mg/mL(3:1). The remaining prototypes Combo 4, Combo 5, Combo 6, and Combo 8were prepared in the same fashion as Combo New, modified to the specificconcentrations shown in Table 4.

The combined DP formulations were then filtered and sterile filled into10 cc vials (SAP #1215125, batch #2L68780), stoppered (SAP #1239068,batch #0H49862), and crimped. Some vials were saved for day-0 controlanalysis. The rest were put on stability stations at 5° C., 25° C., and40° C. until vials were pulled at specific time points for analysis.

SEC Analysis

SEC was performed on the nivolumab DP control, the ipilimumab DPcontrol, and the DoE FDRC (3:1) formulations Combo New, Combo 4, Combo5, Combo 6, and Combo 8 following storage for 3 months at 40° C. (FIG.5A). The ipilimumab control formulation had an initial HMW peak size ofabout 0.4% at day 0, which increased by about 0.2% to a final HMW peaksize of about 0.6% after 3 months at 40° C. (FIG. 5A). The nivolumabcontrol formulation had an initial HMW peak size of about 0.7% at day 0,which increased by about 1.6% to a final HMW peak size of about 2.4%after 3 months at 40° C. (FIG. 5A). The Combo New FDRC formulation hadan initial HMW peak size of about 0.4% at day 0, which increased byabout 0.1% to a final HMW peak size of just over 0.5% after 3 months at40° C. (FIG. 5A). The Combo 4 FDRC formulation had an initial HMW peaksize of about 0.6% at day 0, which increased by about 0.7% to a finalHMW peak size of about 1.3% after 3 months at 40° C. (FIG. 5A). TheCombo 5 FDRC formulation had an initial HMW peak size of just less than0.5% at day 0, which increased by about 0.3% to a final HMW peak size ofless than 0.8% after 3 months at 40° C. (FIG. 5A). The Combo 6 FDRCformulation had an initial HMW peak size of about 0.5% at day 0, whichincreased by about 0.3% to a final HMW peak size of about 0.8% after 3months at 40° C. (FIG. 5A). The Combo 8 FDRC formulation had an initialHMW peak size of about 0.5% at day 0, which increased by about 1.0% to afinal HMW peak size of about 1.5% after 3 months at 40° C. (FIG. 5A).

The same formulations were analyzed by SEC following storage for 3months at 25° C. (FIG. 5A). The HMW peak size of the ipilimumab controlformulation and the nivolumab control formulation each increased 0.1% orless following storage for 3 months at 25° C. (FIG. 5A). The HMW peaksizes of the Combo New and Combo 8 FDRC formulations each increased by0.1% or less, and the HMW peak sizes of the Combo 4, Combo 5, and Combo6 FDRC formulations each decreased by about 0.1% or less followingstorage for 3 months at 25° C. (FIG. 5A).

cIEF Analysis

cIEF was performed on the nivolumab DP control, the ipilimumab DPcontrol, and the DoE FDRC (3:1) formulations Combo New, Combo 4, Combo5, Combo 6, and Combo 8 following storage for 3 months at 25° C. (FIG.5B). The ipilimumab control acidic peak size increased by about 5.59%,and the nivolumab DP control acidic peak size decreased by about 0.05%following storage for 3 months at 25° C. (FIG. 5B). The nivolumab andipilimumab acidic peak sizes of the Combo New FDRC formulation increasedby about 0.64% and 5.98%, respectively, following storage for 3 monthsat 25° C. (FIG. 5B). The nivolumab and ipilimumab acidic peak sizes ofthe Combo 4 FDRC formulation increased by about 5.32% and 6.97%,respectively, following storage for 3 months at 25° C. (FIG. 5B). Thenivolumab and ipilimumab acidic peak sizes of the Combo 5 FDRCformulation increased by about 0.12% and 5.34%, respectively, followingstorage for 3 months at 25° C. (FIG. 5B). The nivolumab and ipilimumabacidic peak sizes of the Combo 6 FDRC formulation increased by about7.01% and 12.19%, respectively, following storage for 3 months at 25° C.(FIG. 5B). The nivolumab and ipilimumab acidic peak sizes of the Combo 8FDRC formulation each increased by about 7.17% following storage for 3months at 25° C. (FIG. 5B).

Example 4

A feasibility study was performed to evaluate the stability of anipilimumab/nivolumab FDRC at varying ratios of ipilimumab to nivolumab,using a modified version of the DoE FDRC (3:1) Combo New formulation,characterized in Example 3, as the base formulation.Ipilimumab/nivolumab FDRC platform combined (PC) formulations were madeat ratios of ipilimumab to nivolumab of 3:1, 1:3, and 1:1, as shown inTable 5. All formulations were prepared in a histidine buffer and atfinal concentrations of 50 μM DTPA, 0.05% w/v PS80, and 8.0% w/v sucrose(Table 5). FDRC PC Prototype 4 (“PC: pH 5.5-1:3”) had a ratio of 1:3 anda pH of 5.5; FDRC PC Prototype 5 (“PC: pH 6.0-1:3”) had a ratio of 1:3and a pH of 6.0; FDRC PC Prototype 6 (“PC: pH 6.5-1:3”) had a ratio of1:3 and pH of 6.5; FDRC PC Prototype 7 (“PC: pH 6.0-1:1”) had a ratio of1:1 and a pH of 6.0; and FDRC PC Prototype 8 (“PC: pH 6.0-3:1”) had aratio of 3:1 and a pH of 6.0 (Table 5).

TABLE 5 Ipilimumab/Nivolumab FDRC Platform Combined Formulations FinalConc'n in Vial: (mg/mL) Mannitol NaCl DTPA PS 80 Sucrose Prototype RatioIpi Nivo pH % w/v mM μM % w/v % w/v 4 1:3 2.5 7.5 5.5 — — 50 0.05 8.0 51:3 2.5 7.5 6.0 — — 50 0.05 8.0 6 1:3 2.5 7.5 6.5 — — 50 0.05 8.0 7 1:15 5 6.0 — — 50 0.05 8.0 8 3:1 7.5 2.5 6.0 — — 50 0.05 8.0SEC Analysis

SEC was performed on the nivolumab DP control, the ipilimumab DPcontrol, and the platform combined (PC) FDRC formulations PC: pH6.0-1:1, PC: pH 5.5-1:3, PC: pH 6.0-1:3, PC: pH 6.5-1:3, and PC: pH6.0-3:1 following storage for 3 months at 40° C. (FIG. 6A). The HMW peaksizes of the nivolumab and ipilimumab control formulations increased byabout 1.7% and 0.25%, respectively, following storage for 3 months at40° C. (FIG. 6A). The HMW peak size of the PC: pH 6.0-1:1 FDRCformulation increased by about 0.5% following storage for 3 months at40° C. (FIG. 6A). The HMW peak size of the PC: pH 5.5-1:3 FDRCformulation increased by about 1.25% following storage for 3 months at40° C. (FIG. 6A). The HMW peak size of the PC: pH 6.0-1:3 FDRCformulation increased by about 0.75% following storage for 3 months at40° C. (FIG. 6A). The HMW peak size of the PC: pH 6.5-1:3 FDRCformulation increased by about 0.1% following storage for 3 months at40° C. (FIG. 6A). The HMW peak size of the PC: pH 6.0-3:1 FDRCformulation increased by about 0.25% following storage for 3 months at40° C. (FIG. 6A).

The same formulations were analyzed by SEC following storage for 3months at 5° C. (FIG. 6B). The nivolumab control formulation had aninitial HMW peak size of about 0.70% at day 0, which increased to afinal HMW peak size of about 0.71% after 3 months at 5° C. (FIG. 6B).The ipilimumab control formulation had an initial HMW peak size of about0.4% at day 0, which did not change after 3 months at 5° C. (FIG. 6B).The PC: pH 6.0-1:1 FDRC formulation had an initial HMW peak size ofabout 0.44% at day 0, which increased to a final HMW peak size of about0.45% after 3 months at 5° C. (FIG. 6B). The PC: pH 5.5-1:3 FDRCformulation had an initial BMW peak size of about 0.47% at day 0, whichincreased to a final HMW peak size of about 0.48% after 3 months at 5°C. (FIG. 6B). The PC: pH 6.0-1:3 FDRC formulation had an initial HMWpeak size of about 0.51% at day 0, which did not change after 3 monthsat 5° C. (FIG. 6B). The PC: pH 6.5-1:3 FDRC formulation had an initialHMW peak size of about 0.56% at day 0, which increased to a final HMWpeak size of about 0.58% after 3 months at 5° C. (FIG. 6B). The PC: pH6.0-3:1 FDRC formulation had an initial HMW peak size of about 0.37% atday 0, which increased to a final HMW peak size of about 0.39% after 3months at 5° C. (FIG. 6B).

cIEF Analysis

cIEF was performed on the nivolumab DP control, the ipilimumab DPcontrol, and the platform combined (PC) FDRC formulations PC: pH6.0-1:1, PC: pH 5.5-1:3, PC: pH 6.0-1:3, PC: pH 6.5-1:3, and PC: pH6.0-3:1 following storage for 3 months at 25° C. (FIG. 7A) and 3 monthsat 5° C. (FIG. 7B).

Following storage for 3 months at 25° C., the nivolumab control acidicpeak size decreased by about 0.05%, and the ipilimumab control acidicpeak size increased by about 5.59% (FIG. 7A). The nivolumab andipilimumab acidic peak sizes of the PC: pH 6.0-1:1 FDRC formulationincreased by about 2.6% and 7%, respectively, following storage for 3months at 25° C. (FIG. 7A). The PC: pH 5.5-1:3 FDRC formulationnivolumab and ipilimumab acidic peak sizes increased by about 2.1% and5.9%, respectively, following storage for 3 months at 25° C. (FIG. 7A).The PC: pH 6.0-1:3 FDRC formulation nivolumab and ipilimumab acidic peaksizes increased by about 3.7% and 6.8%, respectively, following storagefor 3 months at 25° C. (FIG. 7A). The PC: pH 6.5-1:3 FDRC formulationnivolumab and ipilimumab acidic peak sizes increased by about 5.9% and6.3%, respectively, following storage for 3 months at 25° C. (FIG. 7A).The PC: pH 6.0-3:1 FDRC formulation nivolumab and ipilimumab acidic peaksizes increased by about 1.3% and 6.2%, respectively, following storagefor 3 months at 25° C. (FIG. 7A). Across all PC FDRC formulations storedfor 3 months at 25° C., the ipilimumab acidic peak size increased byabout 5.9-7.0%, or by an average of about 2.0% per month (FIG. 7A). Thenivolumab acidic peak size of the FC FDRC formulations increased byabout 1.3-5.9%, or at a maximum of about 2% per month (FIG. 7A).

Following storage for 3 months at 5° C., the nivolumab control acidicpeak size decreased by about 5.2%, and the ipilimumab control acidicpeak size decreased by about 1% (FIG. 7B). The nivolumab and ipilimumabacidic peak sizes of the PC: pH 6.0-1:1 FDRC formulation decreased byabout 2% and increased by about 2.2%, respectively, following storagefor 3 months at 5° C. (FIG. 7B). The nivolumab and ipilimumab acidicpeak sizes of the PC: pH 5.5-1:3 FDRC formulation decreased by about1.1% and about 0.3%, respectively, following storage for 3 months at 5°C. (FIG. 7B). The nivolumab and ipilimumab acidic peak sizes of the PC:pH 6.0-1:3 FDRC formulation each decreased by about 0.2% followingstorage for 3 months at 5° C. (FIG. 7B). The nivolumab and ipilimumabacidic peak sizes of the PC: pH 6.5-1:3 FDRC formulation increased byabout 0.5% and decreased by about 3.1%, respectively, following storagefor 3 months at 5° C. (FIG. 7B). The nivolumab and ipilimumab acidicpeak sizes of the PC: pH 6.0-3:1 FDRC formulation increased by about0.1% and decreased by about 0.2%, respectively, following storage for 3months at 5° C. (FIG. 7B). In sum, the acidic peak sizes of ipilimumaband nivolumab in the 1:3 formulations across pH 5.5-6.5 showedessentially no change after storage for 3 months at 5° C., and there wasno discernable change in ipilimumab and nivolumab across the 3 differentratios (FIG. 7B).

Example 5

A feasibility study was performed to evaluate the stability of anipilimumab/nivolumab (1:1) FDRC in several nivolumab-DP-basedformulations, as shown in Table 6. These formulations were designedthrough modification of the nivolumab DP formulation (FIG. 1). A totalof 24 vials of ipilimumab DP and nivolumab DP were subjected to bufferexchange from their original DP buffer formulations into a bufferformulation containing 20 mM citric acid and 50 mM NaCl at pH 6.0(Prototype A) using centrifugal filter units with a molecular weightcutoff of 50 kDa. Prototypes B-D were prepared in the same manner toreach the specifications shown in Table 6. Prototype A contained 7.5mg/mL ipilimumab, 7.5 mg/mL nivolumab, 20 mM citrate, 50 mM NaCl, 3.0%w/v mannitol, 100 μM pentetic acid (DTPA), and 0.02% PS80, at pH 6.0.Prototype A was identical to the nivolumab DP except that Prototype Ahad 100 μM pentetic acid, whereas the nivolumab DP had 20 μM penteticacid. Prototype B contained 7.5 mg/mL ipilimumab, 7.5 mg/mL nivolumab,20 mM citrate, 50 mM NaCl, 3.0% w/v mannitol, 100 μM pentetic acid(DTPA), and 0.02% PS80, at pH 6.5. Prototype C contained 7.5 mg/mLipilimumab, 7.5 mg/mL nivolumab, 20 mM citrate, 100 mM NaCl, 1.0% w/vmannitol, 100 μM pentetic acid (DTPA), and 0.02% PS80, at pH 6.5.Prototype D contained 7.5 mg/mL ipilimumab, 7.5 mg/mL nivolumab, 20 mMcitrate, 50 mM NaCl, 6% w/v sucrose, 100 μM pentetic acid (DTPA), and0.02% PS80, at pH 6.0.

TABLE 6 Nivolumab-DP-based FDRC (1:1) Formulations Prototype A:Ipi/Nivo: 7.5 + 7.5 in Citrate (20 mM), pH 6.0, 50 mM NaCl, 3.0% w/vMannitol, 100 uM Pentetic acid, 0.02% PS80 Prototype B: Ipi/Nivo: 7.5 +7.5 in Citrate (20 mM), pH 6.5, 50 mM NaCl, 3.0% w/v Mannitol, 100 uMPentetic acid, 0.02% PS80 Prototype C: Ipi/Nivo: 7.5 + 7.5 in Citrate(20 mM), pH 6.5, 100 mM NaCl, 1.0% w/v Mannitol, 100 uM Pentetic acid,0.02% PS80 Prototype D: Ipi/Nivo: 7.5 + 7.5 in Citrate (20 mM), pH 6.0,50 mM NaCl, 6% w/v Sucrose, 100 uM Pentetic acid, 0.02% PS80 * Note:Prototype A is similar to the nivolumab DP formulation except thepentetic acid (DTPA) concentration is the same as in the ipilimumab DPformulation (see FIG. 1).

FDRC prototypes A, B, C, and D were filtered with 0.2 micron unit andfilled into 10 cc SCHOTT® vials (1 or 2 mL per vial), stoppered, andsealed. They were then put on stability stations for up to 12 months forstability analysis by appearance, pH, SEC, HIAC, and cIEF.

SEC Analysis

SEC was performed on the nivolumab DP control, the ipilimumab DPcontrol, and the nivolumab-DP-based FDRC (1:1) prototypes A, B, C, and Dfollowing storage for 1 month at 40° C. (FIG. 8). The BMW peak sizes ofthe nivolumab and ipilimumab control formulations increased by about0.38% and 0.02%, respectively, following storage for 1 month at 40° C.(FIG. 8). The HMW peak size of the FDRC prototype A formulationincreased by about 0.36% following storage for 1 month at 40° C. (FIG.8). The HMW peak size of the FDRC prototype B formulation increased byabout 0.41% following storage for 1 month at 40° C. (FIG. 8). The HMWpeak size of the FDRC prototype C increased by about 0.37% followingstorage for 1 month at 40° C. (FIG. 8). The HMW peak size of the FDRCprototype D increased by about 0.24% following storage for 1 month at40° C. (FIG. 8). The nivolumab control formulation and FDRC prototype Aand B formulations each contained 3% w/v mannitol, whereas theipilimumab control formulation and FDRC prototype C formulation had 1%mannitol and the FDRC prototype D formulation had no mannitol (see Table6).

cIEF Analysis

cIEF was performed on the nivolumab DP control, the ipilimumab DPcontrol, and the nivolumab-DP-based FDRC (1:1) prototype A, B, C, and Dformulations following storage for 3 months at 25° C. (FIG. 9). Thenivolumab control acidic peak size increased by about 7.5%, and theipilimumab control acidic peak size increased by about 8.8% followingstorage for 3 months at 25° C. (FIG. 9). The nivolumab and ipilimumabacidic peak sizes of the FDRC prototype A formulation each increased byabout 9.4% following storage for 3 months at 25° C. (FIG. 9). Thenivolumab and ipilimumab acidic peak sizes of the FDRC prototype Bformulation increased by about 8.2% and 13.8%, respectively, followingstorage for 3 months at 25° C. (FIG. 9). The nivolumab and ipilimumabacidic peak sizes of the FDRC prototype C formulation increased by about8.7% and 10.2%, respectively, following storage for 3 months at 25° C.(FIG. 9). The nivolumab and ipilimumab acidic peak sizes of the FDRCprototype D formulation increased by about 10.1% and 9%, respectively,following storage for 3 months at 25° C. (FIG. 9). The effect of NaCl onacidic peak change can be observed by comparing the ipilimumab controlformulation and the FDRC prototype C formulation, which each had 100 mMNaCl, to the nivolumab control formulation and the FDRC prototype A, B,and D formulations, which each had 50 mM NaCl (FIG. 9; Table 6).

Example 6

A fixed dose ratio combination (FDRC) drug product of Nivolumab andIpilimumab was developed in 1:3 ratio. Ipilimumab/nivolumab FDRC wasprepared from the commercial drug substance of ipilimumab and nivolumab.See FIG. 1. Ipilimumab drug substance is an aqueous solution containing5 mg/mL ipilimumab in 20 mM Tris hydrochloride, 100 mM sodium chloride,1.0% (w/v) mannitol, 100 μM pentetic acid, 0.01% (w/v) polysorbate 80 atpH 7.0. Nivolumab drug substance is an aqueous solution containing 20mg/mL nivolumab in 20 mM sodium citrate, 50 mM sodium chloride, 3.0%(w/v) mannitol, 20 μM pentetic acid, 0.04% (w/v) polysorbate 80 at pH6.0. Both the ipilimumab and nivolumab drug substance are stored at2°-8° C.

The ipilimumab/nivolumab FDRC (3:1) drug product is formulated bycombining the ipilimumab and nivolumab drug substances at an ipilimumabto nivolumab protein ratio of 3 to 1. Development stability data up to 6months showed that the FDRC drug product was stable when stored at 2° to8° C., the intended storage condition. The FDRC drug product is asterile, non-pyrogenic, single-use, preservative-free, isotonic aqueoussolution for IV administration. The FDRC drug product may beadministered undiluted at a total protein concentration of 6.2 mg/mL orfurther diluted with 0.9% Sodium Chloride Injection, USP or 5% DextroseInjection, USP to desired concentrations. The FDRC is packaged in a TypeI flint glass tubing or molded vial and stoppered with a FLUROTEC®film-coated butyl rubber stopper. The composition of the FDRC isprovided in Table 7.

TABLE 7 Comparison of the Compositions of the FDRC, Ipilimumab, andNivolumab Drug Products Quantity (Concentration) Nivo- ComponentFunction FDRC Ipilimumab lumab Unit Ipilimumab Active 4.62 5 — mg/mL(BMS-734016) ingredient Nivolumab Active 1.54 — 10 mg/mL (BMS-936558)ingredient Tris Buffering 18.5 20 — mM Hydrochloride agent SodiumCitrate, Buffering 1.5 — 20 mM Dihydrate agent Sodium Tonicity 96.2 10050 mM Chloride modifier Mannitol Tonicity 1.2 1.0 3.0 % (w/v) modifierPentetic Acid¹ Metal ion 93.9 100.0 20.0 μM chelator Polysorbate 80Surfactant 0.012 0.010 0.020 % (w/v) pH at 20° to pH 6.0 7.0 (at 4° C.)6.0 pH unit 25° C.² adjustment Water for Solvent q.s. Injection ¹Alsoknown as diethylenetriaminepentaacetic acid (DTPA) ²Diluted solutions ofhydrochloric acid and sodium hydroxide may be used for pH adjustmentduring ipilimumab and nivolumab DS manufacturing. Solution pH is notadjusted during DP manufacturing

The stability of the FDRC DP samples prepared as shown in Table 7 wasmonitored under intended (5° C.), accelerated (25° C.), and stressed(40° C.) storage conditions.

The major degradation pathways of ipilimumab and nivolumab wereidentified to be aggregation (HMW species detected by SE-HPLC),fragmentation (LMW species detected by SE-HPLC), and deamidation (acidiccharge variants detected by CEX or iCIEF) as shown in Examples 1 to 5.These changes were monitored by SE-HPLC and iCIEF in the FDRCdevelopment stability study. In addition, the particulate matter andbinding activities were monitored by HIAC and ELISA binding,respectively, in these studies at designated time points.

The results obtained from the studies showed that in the FDRC DP, thelevels of combined HMW species, combined LMW species, acidic chargevariants of nivolumab, acidic charge variants of ipilimumab, andparticulate matter remained essentially unchanged after 6-months storageat 2° C. to 8° C.

The studies conducted through 6 months under the accelerated conditionof 25° C. showed that the rate of formation of HMW species is comparablebetween the FDRC DP, nivolumab DP, and ipilimumab DP. The rate offormation of LMW species in the FDRC DP is 0.15% per month, which iscomparable with that of 0.18% per month in the ipilimumab DP, as theFDRC is primarily composed of ipilimumab. The rate of formation ofnivolumab acidic variants in the FDRC DP is 1.98% per month, which iscomparable with that of 1.76% per month in the nivolumab DP. The rate offormation of ipilimumab acidic variants in the FDRC and in theipilimumab DP is considered comparable at 2.4% and 1.9% per month,respectively. The level of particulate matter remained essentiallyunchanged.

The studies conducted through 3-months storage at 40° C. showed thatsimilar but greater changes were observed in the FDRC DP under thecondition.

The use-time study data demonstrate the stability, compatibility, andequivalence of the dosing solutions prepared from the FDRC DP and fromcombining individual nivolumab and ipilimumab DPs in an IV bag.

In summary, comparable formation rates of critical quality attributes(CQAs) such as HMW species, LMW species, and acidic variants at stressedand accelerated conditions and negligible changes in these CQAs at therecommended storage condition indicate the potential for developing aFDRC DP with the commercialized DS of nivolumab and ipilimumab. Each ofthe above listed studies are shown in more detail below:

Aggregation and Fragmentation Detected by SE-HPLC

The extent of aggregation (HMW species) and fragmentation (LMW species)of the FDRC was examined by SE-HPLC. The HMW species, monomer, and LMWspecies of ipilimumab co-elute with the HMW species, monomer, and LMWspecies of nivolumab, respectively. The results presented in Table 8 arereported as the area percent of combined monomer, combined HMW species,and combined LMW species of ipilimumab and nivolumab. The level ofcombined HMW species was tested 0.5% initially and remained essentiallyunchanged through 6 months of storage at 5° C. and 25° C. (ranged 0.5 to0.6%) and increased to 1.0% through 3 months of storage at 40° C. Thelevel of combined LMW species was tested 0.1% initially and remainedessentially unchanged through 6 months of storage at 5° C. (ranged 0.1to 0.2%), increased to 1.0% through 6 months of storage at 25° C., andincreased to 2.4% through 3 months of storage at 40° C.

TABLE 8 Combined Monomer, HMW Species, and LMW Species in the FDRCDetected by SE-HPLC Combined Monomer Combined HMW Combined LMW Time (%)Species (%) Species (%) (Month) 5° C. 25° C. 40° C. 5° C. 25° C. 40° C.5° C. 25° C. 40° C. Initial 99.3 99.3 99.3 0.5 0.5 0.5 0.1 0.1 0.1 1 NT99.3 98.9 NT 0.6 0.6 NT 0.2 0.4 2 99.3 99.2 97.5 0.5 0.5 0.8 0.2 0.3 1.73 99.3 98.8 96.7 0.5 0.6 1.0 0.2 0.6 2.4 6 99.3 98.4 NT 0.6 0.7 NT 0.21.0 NT NT = not tested

These results were compared with ipilimumab (5 mg/mL) and nivolumab (10mg/mL) commercial DP formulation controls placed on station at 5° C.,25° C. and 40° C. along with FDRC DP analyzed in a similar manner withthis modified SEC-HPLC method as shown in Table 9 and 10 respectively.Based on the data available it is evident that both ipilimumab,nivolumab and FDRC are not prone to formation of HMW species at therecommended storage temperature of 2-8° C.; and the rate of formation ofHMW species/month is comparable between ipilimumab, nivolumab and FDRCat 25° C. and 40° C. conditions as shown in Table 11. More importantly,the rates of formation of HMW and LMW species in FDRC are equivalent torates of formation of HMW & LMW species in ipilimumab, as the FDRC ispredominantly composed of ipilimumab and the total protein concentrationin FDRC i.e., 6.2 mg/mL is very close to ipilimumab DP concentration of5 mg/mL. Comparable formation rates of CQA's such as HMW and LMW speciesat stressed and accelerated conditions and negligible change in theseCQA's at recommended storage conditions indicate the potential for adevelopment for FDRC DP.

TABLE 9 Monomer, HMW Species, and LMW Species in Ipilimumab Detected bySE-HPLC Time Monomer (%) HMW Species (%) LMW Species (%) (Month) 5° C.25° C. 40° C. 5° C. 25° C. 40° C. 5° C. 25° C. 40° C. Initial 99.3899.38 99.38 0.4 0.4 0.4 0.22 0.22 0.22 1 99.37 99.33 98.98 0.4 0.38 0.420.23 0.29 0.6 2 99.39 99.26 98.54 0.39 0.395 0.51 0.22 0.325 0.955 399.37 99.16 96.65 0.4 0.43 0.635 0.23 0.415 2.54 6 99.29 98.62 NT 0.4750.535 NT 0.245 1.32 NT

TABLE 10 Monomer, HMW Species, and LMW Species in Nivolumab Detected bySE-HPLC Time Monomer (%) HMW Species (%) LMW Species (%) (Month) 5° C.25° C. 40° C. 5° C. 25° C. 40° C. 5° C. 25° C. 40° C. Initial 98.9998.99 98.99 0.705 0.705 0.705 0.315 0.315 0.315 1 98.98 98.94 98.350.705 0.73 1.115 0.32 0.33 0.55 2 98.98 98.91 97.56 0.705 0.76 1.5950.305 0.34 0.81 3 98.98 98.79 96.45 0.715 0.815 2.37 0.31 0.395 1.18 698.91 98.14 NT 0.795 0.965 NT 0.3 0.425 NT

TABLE 11 Rate of formation/month of Monomer, HMW Species, and LMWSpecies in Ipilimumab, Nivolumab and FDRC as detected by SEHPLC TimeMonomer (%) HMW Species (%) LMW Species (%) (Month) 5° C. 25° C. 40° C.5° C. 25° C. 40° C. 5° C. 25° C. 40° C. FDRC 0.00 0.16 0.92 0.02 0.030.17 0.02 0.15 0.82 Ipilimumab 0.01 0.13 0.86 0.01 0.02 0.08 0.00 0.180.73 Nivolumab 0.01 0.14 0.84 0.02 0.04 0.55 0.00 0.02 0.29Charge Variants Detected by iCIEF

The charge variant profile of the FDRC was determined by iCIEF analysis.Ipilimumab and nivolumab peaks are separated in the chromatographicprofile. The relative amount of the acidic peak areas, main peak area,and basic peak areas of ipilimumab are provided in Table 12 and therelative amount of the acidic peak areas, main peak area, and basic peakareas of nivolumab are provided in Table 13. The acidic, main, and basicpeak areas of both ipilimumab and nivolumab remained essentiallyunchanged through 6 months of storage at 5° C. Changes to the chargeprofile were observed at 25 C and 40 C for both ipilimumab andnivolumab. Degradation was significant at 40° C. within a very shortduration and hence not used for comparison and considered too aggressivefor evaluation of DP stability.

TABLE 12 Charge Profile of Ipilimumab in the FDRC Detected by iCIEF TimeAcidic Peaks (%) Main Peak (%) Basic Peaks (%) (Month) 5° C. 25° C. 40°C. 5° C. 25° C. 40° C. 5° C. 25° C. 40° C. Initial 34.6 34.6 34.6 59.559.5 59.5 5.9 5.9 5.9 1 NT 37.2 48.7 NT 56.7 45.2 NT 6.2 6.1 2 34.5 39.661.2 57.9 54.5 33.7 7.6 5.9 5.1 3 34.6 42.4 68.3 59.2 51.9 27.4 6.2 5.74.3 6 35.6 49.0 NT 59.0 45.9 NT 5.4 5.1 NT

TABLE 13 Charge Profile of Nivolumab in the FDRC Detected by iCIEF TimeAcidic Peaks (%) Main Peak (%) Basic Peaks (%) (Month) 5° C. 25° C. 40°C. 5° C. 25° C. 40° C. 5° C. 25° C. 40° C. Initial 33.7 33.7 33.7 59.859.8 59.8 6.5 6.5 6.5 1 NT 34.3 45.0 NT 59.8 46.3 NT 5.9 8.7 2 34.8 39.161.3 59.3 54.5 31.2 5.9 6.5 7.5 3 33.6 37.7 65.9 60.6 55.2 27.9 5.9 7.16.2 6 33.7 45.5 NT 59.3 46.4 NT 7.1 8.1 NT

Moreover, as discussed earlier the FDRC composition evaluated above iscomposed of tris.HCl and sodium citrate dihydrate, and hence prone to pHchanges with temperature because of the amine buffer Tris-HCl. Hence,charge profile changes between FDRC and nivolumab were conducted at 25°C., to be consistent with sample preparation temperature and storagetemperature.

Comparison of ipilimumab acidic charge profile in FDRC shown in Table 12against ipilimumab control DP in Tris-HCl buffer (commercialcomposition) at pH 7 (4° C.) at 25° C. condition (Table 14) hasindicated that the acidic peak formation is comparable at 2.4% and1.93%/month respectively as shown in FIG. 10. This relatively minordifference at stressed conditions is considered insignificant for theFDRC drug product stability at the recommended storage conditions (2-8°C.) as observed in Table 12.

Historically, changes in ipilimumab charge profile have been monitoredby CEX and hence data is being collected to identify the comparabilityof the charge profile at various conditions for ipilimumab, however, thepotential for deamidation, the primary degradation pathway foripilimumab, is potentially decreased since deamidation kinetics areusually slower at lower pH.

TABLE 14 Charge Profile of Ipilimumab by iCIEF at 25 C. Acidic Peaks (%)Main Peak (%) Basic Peaks (%) Time Lab Stability Lab Stability LabStability (Month) Batch Batch Batch Initial 39.71 56.59 3.69 1 42.3454.84 2.82 3 45.3 51.2 3.5 6 51.6 45 3.4

Comparison of nivolumab acidic charge profile in FDRC shown in Table 15against three nivolumab long term stability batches (LTSB) at 25° C.condition has indicated that the acidic peak formation is comparable at1.97% and 1.75%/month respectively as shown in FIG. 11.

TABLE 15 Charge Profile of Nivolumab (in GMP Batches) by iCIEF at 25° C.Acidic Peaks (%) Main Peak (%) Basic Peaks (%) Time Lot Lot Lot Lot LotLot Lot Lot Lot (Month) 2J71008 2J71191 2J71192 2J71008 2J71191 2J711922J71008 2J71191 2J71192 Initial 36.1 36.3 35.2 58.1 56.6 58.8 5.8 7.16.0 1 33.0 29.6 36.2 61.8 62.9 57.2 5.2 7.5 6.6 3 40.8 39.3 37.2 52.952.4 55.6 6.3 8.3 7.2 6 45.7 44.1 44.3 46.4 47.2 47.8 7.9 8.7 7.9Particulate Matter Detected by HIAC

Samples stored at 5° and 25° C. for up to 6 months were examined usinglight obscuration particle count procedure (HIAC) to determine the sizeand number of particles according to size in the FDRC DP. As shown inTable 16, particulate matter values for ≥2 microns, ≥5 microns, ≥10microns, and ≥25 microns were variable, but were well within theacceptance criteria outlined in USP <787>.

TABLE 16 Particulate Matter by HIAC Particulate Matter (Particles/mL) ≥2μm ≥5 μm ≥10 μm ≥25 μm Initial 161 21 5 0 6 Months at 5° C. 134 43 13 26 Months at 25° C. 174 34 6 1Binding Activity Determined by ELISA Assays

ELISA assays were utilized to examine the specific binding of ipilimumabto human CTLA-4 receptor and the specific binding of nivolumab to humanPD-1 receptor. The binding activities of ipilimumab and nivolumab in theFDRC samples were calculated relative to the ipilimumab and nivolumabreference standards, respectively. The binding activities of the FDRCsamples through 2 months of storage at 25° C. were within the proposedacceptance criterion (70 to 130%) (Table 17).

TABLE 17 Binding Activities Determined by ELISA Assays IpilimumabBinding Nivolumab Binding Activity Relative to Activity Relative toStorage Ipilimumab Reference Nivolumab Reference Condition Standard (%)Standard (%) Initial 87 119 2 Months at 87 78 25° C.Tryptic Peptide Mapping Assay

A tryptic peptide mapping assay was performed to measure deamidation andoxidation. Samples were reduced, alkylated and digested with trypsin.The tryptic peptides were separated on a C-18 column and detected by aUV detector at 215 and 280 nm, followed by a mass spectrometer(LTQ-Orbitrap-Elite). Relative quantitation was achieved by comparingpeak areas of the intact peptides as well as the modified peptides inselected ion chromatograms. The results of the assay are shown in Tables18 and 19.

TABLE 18 Tryptic Peptide Mapping - Deamidation Ipi Ipi Nivo Ipi H37/NivoIpi H37/Nivo H37/Nivo H37/Nivo Storage H₄ ³ Ipi H₅ ⁴ H36 Deam1⁵ H36Deam2⁶ H36 Deam3⁷ H36 Deam4⁸ Condition (%) (%) (%) (%) (%) (%) Initial0.5 2.8 3.4 3.2 1.4 3.6 2 Months at 25° C. 0.7 4.6 4.7 4.6 1.5 3.7 6Months at 25° C. 1.0 3.9 5.2 5.9 0.7 2.5 ³Nivo H₄ = Heavy Chain TrypticPeptide # 4 for nivolumab ⁴Ipi H₅ = Heavy Chain Tryptic Peptide # 5 foripilimumab ⁵Ipi H₃₇/Nivo H₃₆ Deam1 = Heavy Chain Tryptic Peptide # 37for ipilimumab (Asn #) and #36 for nivolumab (Asn #) ⁶Ipi H₃₇/Nivo H₃₆Deam2 = Heavy Chain Tryptic Peptide # 37 for ipilimumab (Asn #) and #36for nivolumab (Asn #) ⁷Ipi H₃₇/Nivo H₃₆ Deam3 = Heavy Chain TrypticPeptide # 37 for ipilimumab (Asn #) and #36 for nivolumab (Asn #) ⁸IpiH₃₇/Nivo H₃₆ Deam4 = Heavy Chain Tryptic Peptide # 37 for ipilimumab(Asn #) and #36 for nivolumab (Asn #)

TABLE 19 Tryptic Peptide Mapping - Oxidation Ipi H₂₁/ Storage ConditionNivo H₂₂ ⁹ (%) Nivo H4¹⁰(%) Ipi H₃ ¹¹(%) Initial 4.4 0.2 0.5 2 Months at25° C. 4.9 0.6 0.5 6 Months at 25° C. 3.3 0.3 0.2 ⁹Ipi H₂₁/Nivo H₂₂ =Heavy Chain Tryptic Peptide # 21 for ipilimumab (His/Met #) and #22 fornivolumab (His/Met #) ¹⁰Nivo H₄ = Heavy Chain Tryptic Peptide # 4 fornivolumab ¹¹Ipi H₃ = Heavy Chain Tryptic Peptide # 3 for ipilimumabpH Analysis of Samples

The pH of the FDCR DP was measured, as seen in Table 20.

TABLE 20 pH of the DP Solutions Storage Condition 25° C. 5° C. Initial6.58 6.58 2 Months 6.59 6.56 6 Months 6.44 6.38Use-Time Stability of the FDRC Drug Product

A study was performed to demonstrate the stability and compatibility ofthe FDRC DP with 0.9% Sodium Chloride Injection, USP (NS), IV bags, IVinfusion sets, and in-line filters. After 2 months of storage at 5° C.,the FDRC DP samples were diluted into NS in an IV bag which was storedat 25° C. for 4 hours followed by 20 hours at 5° C. The solution in theIV bag was then infused through an IV set and an in-line filter. Sampleswere collected and analyzed by HIAC, micro flow imaging (MFI), SE-HPLC,CE-SDS, iCIEF, and reverse phase ultra performance liquid chromatography(RP-UPLC).

The results of the study are presented in Tables 21-23. The data showlittle or no change from the initial values for particulate matter (byHIAC), aggregation (by SE-HPLC), fragmentation (by (SE-HPLC), purity (byCE-SDS), charge variant profile (by iCIEF), and ipilimumab/nivolumabprotein ratio (by RP-UPLC) after completion of the compatibility study.

The results indicate that the FDRC DP can be diluted with 0.9% SodiumChloride Injection, USP to a concentration range of 1.5/0.5 to 4.2/1.4mg/mL ipilimumab/nivolumab for IV infusion. The diluted solution in anIV bag may be stored at 5° C. for up to 24 hours and up to 4 hours ofthe 24 hours may be at room temperature (25° C.).

TABLE 21 Use-Time Stability and Comparability of the FDRC Drug ProductHIAC MFI CE-SDS (Particles/mL) (Particles/mL) SE-HPL (Reduced) ≥2 ≥5 ≥10≥25 2-10 ≥10 ≥25 HMW Monomer LMW Purity Condition μm μm μm μm μm μm μm(%) (%) (%) (%) 1.5/0.5 mg/mL ipilimumab/nivolumab Initial¹² 138 26 5 0353 11.5 0 0.4 99.4 0.3 99.6 24 hours¹³ 213 52 14 0 414 18 1.6 0.3 99.30.4 99.6 4.2/1.4 mg/mL ipilimumab/nivolumab Initial 345 77 24 1 888 6.50 0.4 99.3 0.3 99.6 24 hours 193 36 12 1 455 0 0 0.4 99.3 0.3 99.6¹²Samples collected from a IV bag at time zero after dilution of theFDRC DP in the IV bag ¹³Samples collected after 24-hours storage andinfusion through an IV set and an in-line filter

TABLE 22 Use-Time Stability and Comparability of the FDRC Drug ProductiCIEF-Ipilimumab iCIEF-Nivolumab RP-UPLC Acidic Main Basic Acidic MainBasic Ipilimumab/ Peaks Peak Peaks Peaks Peak Peaks Nivolumab Condition(%) (%) (%) (%) (%) (%) Protein Ratio 1.5/0.5 mg/mL ipilimumab/nivolumabInitial 38.9 56.2 4.9 34.3 57.5 8.3 2.8 24 hours 38.0 56.8 5.1 34.2 57.78.1 2.8 4.2/1.4 mg/mL ipilimumab/nivolumab Initial 37.2 58.1 4.7 34.258.0 7.8 2.9 24 hours 37.9 57.2 4.9 34.8 57.9 7.3 2.8

TABLE 23 Use-Time Stability and Comparability of the FDRC Drug ProductCondition A280 (Traditional) A280 (Solo-VPE) pH 1.5/0.5 mg/mLipilimumab/nivolumab Initial 2.07 1.998 6.36 24 hours 1.924 2.012 6.334.2/1.4 mg/mL ipilimumab/nivolumab Initial 5.618 5.615 6.38 24 hours5.529 5.647 6.37Use-Time Stability of the Co-Administered Ipilimumab and Nivolumab DrugProduct

A study was performed to demonstrate the stability and compatibility ofthe co-administered DP with 0.9% Sodium Chloride Injection, USP (NS), IVbags, IV infusion sets, and in-line filters. Ipilimumab and nivolumabmonotherapy DP vials were diluted into NS in an IV bag which was storedat 25° C. for 4 hours followed by 20 hours at 5° C. The solution in theIV bag was then infused through an IV set and an in-line filter. Sampleswere collected and analyzed by HIAC, micro flow imaging (WI), SE-HPLC,CE-SDS, iCIEF, and reverse phase ultra performance liquid chromatography(RP-UPLC).

The results of the study are presented in Tables 24-26. The data showlittle or no change from the initial values for particulate matter (byHIAC), aggregation (by SE-HPLC), fragmentation (by SE-HPLC), purity (byCE-SDS), charge variant profile (by iCIEF), and ipilimumab/nivolumabprotein ratio (by RP-UPLC) after completion of the compatibility study.The results indicate that the co-administered DP can be diluted with0.9% Sodium Chloride Injection, USP to a concentration range of 1.5/0.5to 4.2/1.4 mg/mL ipilimumab/nivolumab for IV infusion. The dilutedsolution in an IV bag may be stored at 5° C. for up to 24 hours and upto 4 hours of the 24 hours may be at room temperature (25° C.).

TABLE 24 Use-Time Stability and Comparability of the Co-administeredIpilimumab and Nivolumab Drug Product HIAC MFI CE-SDS (Particles/mL)(Particles/mL) SE-HPLC (Reduced) ≥2 ≥5 ≥10 ≥25 2-10 ≥10 ≥25 HMW MonomerLMW Purity Condition μm μm μm μm μm μm μm (%) (%) (%) (%) 1.5/0.5 mg/mLipilimumab/nivolumab (Non-DEHP Intravia bags) Initial¹⁴ 327 75 10 0 10788 1 0.3 99.4 0.2 99.6 24 hours¹⁵ 49 9 1 0 152 3 1 0.3 99.4 0.3 99.61.5/0.5 mg/mL ipilimumab/nivolumab (DEHP Viaflex bags) Initial 373 54 80 1233 2 0 0.4 99.3 0.3 99.6 24 hours 35 5 1 0 71 5 1 0.4 99.4 0.3 99.64.2/1.4 mg/mL ipilimumab/nivolumab (Non-DEHP Intravia bags) Initial 862170 17 0 2979 8 1 0.4 99.3 0.3 99.6 24 hours 112 21 3 0 237 8 0 0.4 99.40.3 99.6 4.2/1.4 mg/mL ipilimumab/nivolumab (DEHP Viaflex bags) Initial431 91 15 0 1400 11 1 0.4 99.4 0.3 99.6 24 hours 53 11 4 0 150 3 1 0.499.4 0.3 99.6 ¹⁴Samples collected from a IV bag at time zero afterdilution of the co-administered DP in the IV bag ¹⁵Samples collectedafter 24-hours storage and infusion through an IV set and an in-linefilter

TABLE 25 Use-Time Stability and Comparability of the Co-administeredIpilimumab and Nivolumab Drug Product iCIEF-Ipilimumab iCIEF-NivolumabRP-UPLC Acidic Main Basic Acidic Main Basic Ipilimumab/ Peaks Peak PeaksPeaks Peak Peaks Nivolumab Condition (%) (%) (%) (%) (%) (%) ProteinRatio 1.5/0.5 mg/mL ipilimumab/nivolumab (Non-DEHP Intravia bags)Initial 36.3 59.9 3.8 35.9 59.1 5.0 2.85 24 hours 36.2 59.9 3.9 33.860.7 5.5 2.80 1.5/0.5 mg/mL ipilimumab/nivolumab (DEHP Viaflex bags)Initial 36.8 59.8 3.4 35.2 58.6 6.2 2.87 24 hours 36.2 60.1 3.7 35.358.3 6.4 2.79 4.2/1.4 mg/mL ipilimumab/nivolumab (Non-DEHP Intraviabags) Initial 36.6 59.8 3.6 34.1 59.8 6.1 2.9  24 Hours 36.9 59.5 3.633.6 60.4 6.0 2.87 4.2/1.4 mg/mL ipilimumab/nivolumab (DEHP Viaflexbags) Initial 36.3 59.9 3.8 36.0 57.3 6.7 2.93 24 hours 36.7 59.5 3.835.2 58.6 6.2 2.92

TABLE 26 Use-Time Stability and Comparability of the Co-administeredIpilimumab and Nivolumab Drug Product Condition A280 (Traditional) A280(Solo-VPE) pH 1.5/0.5 mg/mL ipilimumab/nivolumab (Non-DEHP Intraviabags) Initial 1.994 1.941 6.21 24 hours 1.898 1.936 6.19 1.5/0.5 mg/mLipilimumab/nivolumab (DEHP Viaflex bags) Initial 2.019 1.982 6.2 24hours 1.879 1.986 6.2 4.2/1.4 mg/mL ipilimumab/nivolumab (Non-DEHPIntravia bags) Initial 5.701 5.481 6.26 24 hours 5.338 5.538 6.244.2/1.4 mg/mL ipilimumab/nivolumab (DEHP Viaflex bags) Initial 5.5865.509 6.26 24 hours 5.363 5.499 6.26

Example 7

The process performance qualification (PPQ) limits for pH andPolysorbate 80 in a Nivolumab-Ipilimumab 1:3 Fixed dose ratiocombination (FDRC) for nivolumab-Ipilimumab 1:3 fixed dose ratiocombination were determined. The quantitative compositions of the FDRCdrug product are seen in Table 27.

TABLE 27 Quantitative Compositions of the FDRC Drug Product Quantity(mg) Per Vial (Nivolumab/Ipilimumab) Component Function Composition30/90 40/120 Nivolumab Active 1.54 mg/mL 31.11 41.58 (BMS-936558)ingredient Ipilimumab Active 4.61 mg/mL 93.32 124.74 (BMS-734016)ingredient Sodium Buffering 1.54 mM 9.15 12.23 Citrate, agent DihydrateTris Buffering 18.46 mM 58.76 78.55 Hydrochloride agent Sodium Tonicity96.15 mM 113.50 151.71 Chloride modifier Mannitol Tonicity 1.15% w/v232.30 310.50 modifier Pentetic Acid¹⁶ Metal ion 93.85 μM 0.75 1.00chelator Polysorbate 80 Surfactant 0.012% w/v 2.42 3.24 Hydrochloric pHq.s. to pH 6.3 Acid Sodium adjustment Hydroxide¹⁷ Water for Solvent q.s.to q.s. to Injection 20.2 mL 27.0 mL q.s. = quantity sufficient ¹⁶Alsoknown as diethylenetriaminepentaacetic acid ¹⁷Diluted solutions ofhydrochloric acid and sodium hydroxide may be used for pH adjustmentduring nivolumab and ipilimumab DS manufacturing. Solution pH is notadjusted during FDRC DP manufacturingpH limit

Commercial ipilimumab DS and nivolumab DS have a pH acceptance criteriaof 6.6-7.6 (4° C.) & 5.5-6.5 respectively. Since, the FDRC DP ismanufactured without any further manipulation to the incoming DS, astudy was conducted to understand the potential range of pH in FDRC DPdue to the incoming DS variability.

TABLE 28 Variability in FDRC DP pH due to incoming DS variability 20 mMSodium Final pH 20 mM Tris-HCl Citrate pH (4° C.) pH (21° C.) pH 6.6 pH5.5 5.77 5.64 pH 7.0 pH 6.0 6.47 6.32 pH 7.6 pH 6.5 7.34 6.99

The solutions were prepared by adding 42 mL of 20 mM Tris-HCl and 3.5 mL20 mM Sodium Citrate buffer to mimic FDRC DP preparation. The results ofthis evaluation (Table 28) indicated that the pH range of FDRC DP couldbe in the range of 5.7-7.0, with a target pH of 6.2-6.3 at ambientconditions. This attribute is well controlled in the incoming DS ofnivolumab and ipilimumab, hence it is unlikely that the pH extremes of5.7 or 7.0 will be ever experienced by the FDRC DP. Additionally, basedon the current knowledge of CQAs of ipilimumab and nivolumab, the riskto FDRC DP quality attributes is expected to be higher at the higher pHrange. Based on this understanding, two additional studies wereinitiated to evaluate the impact of pH on DP quality attributes and alsoto understand the impact of variability in various excipients (includingpH) from incoming DS on DP quality attributes.

Evaluation of the data from the pH ranging study was focused on thequality attributes in FDRC impacted by variation in pH such as chargeprofile monitored by capillary isoelectric focusing (icIEF) and highmolecular weight aggregates monitored by size exclusion chromatography(SEC). There is no discernible change in SEC profile of FDRC DP acrossthe pH range of 5.4-6.6 (ambient) at the recommended storage conditionsof 2-8° C. or up to 3 months at 25° C. Quantifiable changes were onlyobserved at accelerated conditions (40° C.), where it was evident thatthe evaluated pH range has no impact on SEC profile (Table 29).

TABLE 29 Monomer, HMW, and LMW Species (%) due to variability in pH DPPrototype HMW Species (%) Monomer (%) LMW Species (%) (pH) Initial 1M/40° C. 3 M/40° C. Initial 1 M/40° C. 3 M/40° C. Initial 1 M/40° C. 3M/40° C. 5.4 1.12 1.25 1.79 98.7 98.2 96.8 0.1 0.53 1.35 5.7 1.19 1.241.69 98.7 98.3 97.1 0.1 0.46 1.16 6.0 1.23 1.24 1.65 98.6 98.3 97.2 0.10.43 1.07 6.3 1.24 1.2 1.63 98.6 98.3 97.3 0.1 0.42 1.04 6.6 1.33 1.331.7 98.6 98.2 97.2 0.1 0.43 1.05

The charge profile of ipilimumab and nivolumab did not shown anysignificant differences beyond analytical error at the recommendedstorage temperature of 2-8° C. after 6 months as shown in Table 30.Charge profile for ipilimumab and nivolumab is primarily evaluated atthe storage temperature of 25° C. as the differences are morediscernible, unlike 40° C., where the profile is dramatically alteredand the molecules significantly fall apart. FIG. 12 illustrates theacidic profile of ipilimumab and nivolumab in FDRC DP in comparison withtheir respective controls at pH 6.0.

TABLE 30 Acidic & Main Peak Profile (cIEF) due to variability in pHafter 6 Months at 5° C. Acidic Peak Profile (%) Main Peak Change (%) (%Change from Initial (% Change from Initial DP Proto- after 6 M at 5° C.)after 6 M at 5° C.) type (pH) Ipilimumab Nivolumab Ipilimumab Nivolumab5.4 −1.0 −0.3 −1.0 −0.4 5.7 0.6 −3.7 1.4 −1.8 6.0 0.6 −1.5 0.8 −1.2 6.30.9 −2.3 1.5 −1.6 6.6 1.1 −1.9 1.6 −0.7

Additionally, a pH ruggedness study initiated with the variables shownin Table 31, where the pH range evaluated was varied between 5.8-7.0 hasresulted in similar observations for SEC and cIEF profiles.

TABLE 31 DP Ruggedness Study Design Final Conc'n in Vial: (mg/mL)Ipilimumab Nivolumab Tris Citrate NaCl Mannitol PS 80 DTPA Prototype(mg/mL) (mg/mL) pH mM mM mM % w/v % w/v μM 1 4.62 1.54 5.8 18.46 1.5476.92 1.38% 0.005 93.85 2 4.62 1.54 5.8 18.46 1.54 115.38 1.38% 0.02093.85 3 4.62 1.54 5.8 18.46 1.54 76.92 0.92% 0.020 93.85 4 4.62 1.54 6.418.46 1.54 96.15 1.15% 0.012 93.85 5 4.62 1.54 7 18.46 1.54 115.38 1.38%0.005 93.85 6 4.62 1.54 7 18.46 1.54 76.92 0.92% 0.005 93.85 7 4.62 1.546.4 18.46 1.54 96.15 1.15% 0.012 93.85 8 4.62 1.54 5.8 18.46 1.54 115.380.92% 0.005 93.85 9 4.62 1.54 7 18.46 1.54 115.38 0.92% 0.020 93.85 104.62 1.54 7 18.46 1.54 76.92 1.38% 0.020 93.85

The HMW profile of the FDRC DP remained unchanged after 6 months ofstorage at 2-8° C. and 25° C. as shown in FIG. 13 (Monomer Profile seenin FIG. 14), indicating the lack of pH effect even in the presence ofother variables such as the concentration of sodium chloride, Mannitoland PS80. Evaluation of acidic and main peak profiles of ipilimumab andnivolumab in FDRC DP (FIG. 15-18) clearly indicates a pH dependence ofdeamidation at accelerated temperature of 25° C., as indicated by anincrease in acidic peak profile at higher temperature range of pH 7.0.This effect at accelerated temperatures, however, does not translateinto a quantifiable difference at the recommended storage temperature of2-8° C.

The cIEF Peak Profile and the impact of pH are shown in FIG. 19, and theiCIEF Profile: pH range of 5.4-6.6 are shown in FIG. 20.

Polysorbate 80 Limit: NLT 60 μg/mL

Polysorbate 80 concentration in the FDRC DP is primarily defined by theproportion of ipilimumab and nivolumab DS mixed to manufacture the FDRCDP, where the target concentration of PS 80 in FDRC DP is 120 μg/mL,with a nominal concentration 100 μg/mL and 400 μg/mL for ipilimumab andnivolumab DS respectively. There are no release acceptance criteria forPS 80 for both nivolumab and ipilimumab DS; however, the nivolumab DSand ipilimumab DS manufacture has an in-process limit of 275-525 μg/mLand 60-140 μg/mL respectively.

Preliminary analysis of the DP attributes that are impacted byvariability of PS80 such as the SEC HMW (%) had no discernible change(FIG. 13) and particulates by HIAC in the range of 10-25 micron range,met the current USP acceptance criteria. Additionally, the FDRC DPmanufacturing process is designed so that there is no need for a filterflush prior to DP vial filling, due to the presence of an intermediatetank (35-40 L) downstream of the redundant sterile filters, which isfilled prior to initiating the filling operation.

Additionally, during DP optimization studies, a PS 80 concentrationrange of 120 μg/ml-1000 μg/ml was evaluated in the FDRC DP under worstcase of agitation at 300 rpm for up to 72 hours on a horizontal shaker.These studies when analyzed by visual appearance showed the lack of anyvisual particulates after 72 hours and the SEC profile for allprototypes had no discernible difference from initial time point. Basedon this, it was decided to not change the target concentration of FDRCDP by spiking with concentrated PS 80 concentration at the DPmanufacturing site.

However, in order to understand the potential risk of particulategeneration or HMW species formation because of significantly lower PS80levels upon dilution with infusion solutions like saline, a study wasconducted where FDRC DP solution with PS 80 at target concentration of120 μg/ml was subjected to 20 fold dilution (6 μg/mL) with normalsaline, and the resulting solutions were evaluated for up to 24 hours byvisual appearance, particulates by HIAC and SEC HMW (%). This studydemonstrated that down to a 6 μg/ml PS 80 concentration in infusionsolutions prepared from FDRC DP does not cause any change in visualappearance, HMW profile or HIAC characteristics of the solution, whichstrengthened the rationale to maintain the target concentration at 120μg/ml. Additionally, it is anticipated that the FDRC DP will be diluted˜3× using infusion solutions, during clinical and commercialadministration which leads to a PS 80 concentration of 40 μg/ml. Theproposed PS 80 concentration of NLT of 60 μg/ml would still result in afinal infusion solution concentration of 20 μg/ml, which is above theevaluated concentration of 6 μg/ml in the dilution study discussedabove.

What is claimed is:
 1. A pharmaceutical composition comprising about1.54 mg/ml nivolumab, about 4.62 mg/ml ipilimumab, about 18.46 mM Trishydrochloride (HCl), about 1.54 mM sodium citrate dihydrate, about 1.15%mannitol, about 96.15 mM NaCl, about 93.85 μM pentetic acid, and about0.012% PS80.
 2. The composition of claim 1, wherein the composition: (i)is stable at about 5° C. for at least about 1 week; (ii) is stable atabout 40° C. for at least about 1 week; or (iii) is stable at about 25°C. for at least about 1 week.
 3. A kit comprising the composition ofclaim 1 and instructions to administer the composition to a subject inneed thereof.
 4. The pharmaceutical composition of claim 1, wherein thepH of the composition is about 6.0, about 6.2, about 6.5, about 6.6, orabout 7.0.
 5. The pharmaceutical composition of claim 1, wherein the pHis about 6.2.
 6. The pharmaceutical composition of claim 1, wherein thepH is about 6.2.
 7. The pharmaceutical composition of claim 1, whereinthe pH is 6.1.
 8. The pharmaceutical composition of claim 1, comprising1.54 mg/mL nivolumab and 4.62 mg/ml ipilimumab.
 9. The pharmaceuticalcomposition of claim 1, comprising about 30 mg nivolumab and about 90 mgipilimumab.
 10. The pharmaceutical composition of claim 1, comprisingabout 40 mg nivolumab and about 120 mg ipilimumab.
 11. Thepharmaceutical composition of claim 1, comprising 18.46 mM Trishydrochloride (HCl).
 12. The pharmaceutical composition of claim 1,comprising 1.54 mM sodium citrate dihydrate.
 13. The pharmaceuticalcomposition of claim 1, comprising 1.15% mannitol.
 14. Thepharmaceutical composition of claim 1, comprising 96.15 mM NaCl.
 15. Thepharmaceutical composition of claim 1, comprising 93.85 μM penteticacid.
 16. The pharmaceutical composition of claim 1, comprising 0.012%PS80.
 17. The pharmaceutical composition of claim 1, comprising 18.46 mMTris hydrochloride (HCl) and 1.54 mM sodium citrate dihydrate.
 18. Thepharmaceutical composition of claim 1, comprising 1.15% mannitol, 96.15mM NaCl, and 93.85 μM pentetic acid.
 19. The pharmaceutical compositionof claim 1, comprising 18.46 mM Tris hydrochloride (HCl), 1.54 mM sodiumcitrate dihydrate, 1.15% mannitol, 96.15 mM NaCl, and 93.85 μM penteticacid.
 20. The pharmaceutical composition of claim 1, comprising 18.46 mMTris hydrochloride (HCl), 1.54 mM sodium citrate dihydrate, 1.15%mannitol, 96.15 mM NaCl, 93.85 μM pentetic acid, and 0.012% PS80. 21.The pharmaceutical composition of claim 20, wherein the pH is 6.2 at 25°C.
 22. The pharmaceutical composition of claim 1, wherein the pH isbetween about 6.0 and about 7.0.
 23. The pharmaceutical composition ofclaim 1, wherein the composition: (i) is stable at 5° C. for at least 1week; (ii) is stable at 40° C. for at least 1 week; or (iii) is stableat 25° C. for at least 1 week.
 24. A pharmaceutical compositioncomprising 1.54 mg/ml nivolumab, 4.62 mg/ml ipilimumab, 18.46 mM Trishydrochloride (HCl), 1.54 mM sodium citrate dihydrate, 1.15% mannitol,96.15 mM NaCl, 93.85 μM pentetic acid, and 0.012% PS80.
 25. Thepharmaceutical composition of claim 24, wherein the pH is between about6.0 and about 7.0.
 26. The pharmaceutical composition of claim 24,wherein the pH is 6.2 at 25° C.
 27. The pharmaceutical composition ofclaim 24, wherein the composition: (i) is stable at 5° C. for at least 1week; (ii) is stable at 40° C. for at least 1 week; or (iii) is stableat 25° C. for at least 1 week.
 28. The pharmaceutical composition ofclaim 24, comprising about 30 mg nivolumab and about 90 mg ipilimumab.29. The pharmaceutical composition of claim 24, comprising about 40 mgnivolumab and about 120 mg ipilimumab.